Material Supplementary 2.DC1http://www.jimmunol.org/content/suppl/2010/06/16/jimmunol.100008
Mucopolysaccharidosis type VI (MPS VI; Maroteaux-Lamy syndrome) is a lysosomal storage disorder caused by mutations in the N-acetylgalactosamine-4-sulfatase (arylsulfatase B, ARSB) gene. ARSB is a lysosomal enzyme involved in the degradation of the glycosaminoglycans (GAG) dermatan and chondroitin sulfate. ARSB mutations reduce enzyme function and GAG degradation, causing lysosomal storage and urinary excretion of these partially degraded substrates. Disease onset and rate of progression is variable, producing a spectrum of clinical presentation. In this study, 105 MPS VI patients-representing about 10% of the world MPS VI population-were studied for molecular genetic and biochemical parameters. Direct sequencing of patient genomic DNA was used to identify ARSB mutations. In total, 83 different disease-causing mutations were found, 62 of which were previously unknown. The novel sequence changes included: 38 missense mutations, five nonsense mutations, 11 deletions, one insertion, seven splice-site mutations, and four polymorphisms. ARSB mutant protein and residual activity were determined on fibroblast extracts for each patient. The identification of many novel mutations unique to individuals/their families highlighted the genetic heterogeneity of the disorder and provided an appropriate cohort to study the MPS VI phenotypic spectrum. This mutation analysis has identified a clear correlation between genotype and urinary GAG that can be used to predict clinical outcome.
Aims/hypothesis Skin lesions and ulcerations are severe complications of diabetes that often result in leg amputations. In this study we investigated the function of the cytoskeletal protein flightless I (FLII) in diabetic wound healing. We hypothesised that overexpression of FLII would have a negative effect on diabetic wound closure and modulation of this protein using specific FLII-neutralising antibodies (FnAb) would enhance cellular proliferation, migration and angiogenesis within the diabetic wound.Methods Using a streptozotocin-induced model of diabetes we investigated the effect of altered FLII levels through Flii genetic knockdown, overexpression or treatment with FnAb on wound healing. Diabetic wounds were assessed using histology, immunohistochemistry and biochemical analysis. In vitro and in vivo assays of angiogenesis were used to assess the angiogenic response. Results FLII levels were elevated in the wounds of both diabetic mice and humans. Reduction in the level of FLII improved healing of murine diabetic wounds and promoted a robust pro-angiogenic response with significantly elevated von Willebrand factor (vWF) and vascular endothelial growth factor (VEGF)-positive endothelial cell infiltration. Diabetic mouse wounds treated intradermally with FnAb showed improved healing and a significantly increased rate of reepithelialisation. FnAb improved the angiogenic response through enhanced formation of capillary tubes and functional neovasculature. Reducing the level of FLII led to increased numbers of mature blood vessels, increased recruitment of smooth muscle actin-α-positive cells and improved tight junction formation. Conclusions/interpretation Reducing the level of FLII in a wound may be a potential therapeutic approach for the treatment of diabetic foot ulcers.
Flightless I (Flii) is an actin remodeling protein that affects cellular processes including adhesion, proliferation and migration. In order to determine the role of Flii during carcinogenesis, squamous cell carcinomas (SCCs) were induced in Flii heterozygous (Flii+/−), wild-type and Flii overexpressing (FliiTg/Tg) mice by intradermal injection of 3-methylcholanthrene (MCA). Flii levels were further assessed in biopsies from human SCCs and the human SCC cell line (MET-1) was used to determine the effect of Flii on cellular invasion. Flii was highly expressed in human SCC biopsies particularly by the invading cells at the tumor edge. FliiTg/Tg mice developed large, aggressive SCCs in response to MCA. In contrast Flii+/− mice had significantly smaller tumors that were less invasive. Intradermal injection of Flii neutralizing antibodies during SCC initiation and progression significantly reduced the size of the tumors and, in vitro, decreased cellular sphere formation and invasion. Analysis of the tumors from the Flii overexpressing mice showed reduced caspase I and annexin V expression suggesting Flii may negatively regulate apoptosis within these tumors. These studies therefore suggest that Flii enhances SCC tumor progression by decreasing apoptosis and enhancing tumor cell invasion. Targeting Flii may be a potential strategy for reducing the severity of SCCs.
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