RESUMO
BACKGROUND: Vasohibin-2 (VASH2) has been identified as an endogenous and vascular endothelial growth factor (VEGF)-independent angiogenic factor that is highly expressed in tumor cells. In the present study, we aimed to determine whether pre-existing vascular changes can be used to predict tumor transformation as benign or malignant. We sought to characterize microvascular changes and tumor development in the intestinal tract of ApcMin/+ mice and ApcMin/+/Vash2-/- mice. METHODS: ApcMin/+ mice provide a unique orthotopic model for the development of spontaneous adenomatous polyposis and subsequent carcinomas, a phenomenon termed the adenoma-carcinoma sequence. ApcMin/+ mice were mated with Vash2-/- mice with a mixed C57BL/6 background and the resulting pups were screened for the Min mutation and for the Vash2-/- gene by PCR. Intestinal tumors from ApcMin/+ mice and ApcMin/+/Vash2-/- mice were removed and either frozen or epon-embedded for subsequent analyses. For 3-dimensional imaging using confocal laser-scanning microscopy and transmission electron microscopy, cryosections were made, and immunofluorescent staining for various markers was performed. RESULTS: We found that structural abnormalities in tumor vessels from benign tumors resembled those in malignant tumors. In addition, a novel angiogenic factor, vasohibin-2 (VASH2) protein, was detected around tumor blood vessels in late-stage adenomas and adenocarcinomas, but was absent from early-stage adenomas in ApcMin/+ mice. Tumors used to examine endogenous VASH2 (derived from CMT93 colon carcinomas) were less vascularized in Vash2-/- mice and were more regular than those seen in wild-type (WT) mice. In addition, tumors in Vash2-/- mice were smaller than those in WT mice. Furthermore, cross-breeding of mice homozygous for a deletion of Vash2 with mice heterozygous for the APC mutation resulted in animals that showed a significant decrease in the number of polyps in the small intestine. CONCLUSION: We propose that VASH2 may modulate the onset of tumors in the gastrointestinal tract by regulating tumor angiogenesis.
Assuntos
Proteína da Polipose Adenomatosa do Colo/genética , Polipose Adenomatosa do Colo/genética , Proteínas Angiogênicas/genética , Trato Gastrointestinal/metabolismo , Regulação Neoplásica da Expressão Gênica , Neovascularização Patológica/prevenção & controle , Polipose Adenomatosa do Colo/metabolismo , Polipose Adenomatosa do Colo/patologia , Proteína da Polipose Adenomatosa do Colo/metabolismo , Proteínas Angiogênicas/metabolismo , Animais , Vasos Sanguíneos/metabolismo , Vasos Sanguíneos/patologia , Carcinogênese/genética , Carcinogênese/metabolismo , Carcinogênese/patologia , Cruzamentos Genéticos , Progressão da Doença , Feminino , Trato Gastrointestinal/irrigação sanguínea , Trato Gastrointestinal/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Transdução de SinaisRESUMO
Trisomy 21 (Down syndrome, DS) is the most common human genetic anomaly associated with heart defects. Based on evolutionary conservation, DS-associated heart defects have been modeled in mice. By generating and analyzing mouse mutants carrying different genomic rearrangements in human chromosome 21 (Hsa21) syntenic regions, we found the triplication of the Tiam1-Kcnj6 region on mouse chromosome 16 (Mmu16) resulted in DS-related cardiovascular abnormalities. In this study, we developed two tandem duplications spanning the Tiam1-Kcnj6 genomic region on Mmu16 using recombinase-mediated genome engineering, Dp(16)3Yey and Dp(16)4Yey, spanning the 2.1 Mb Tiam1-Il10rb and 3.7 Mb Ifnar1-Kcnj6 regions, respectively. We found that Dp(16)4Yey/+, but not Dp(16)3Yey/+, led to heart defects, suggesting the triplication of the Ifnar1-Kcnj6 region is sufficient to cause DS-associated heart defects. Our transcriptional analysis of Dp(16)4Yey/+ embryos showed that the Hsa21 gene orthologs located within the duplicated interval were expressed at the elevated levels, reflecting the consequences of the gene dosage alterations. Therefore, we have identified a 3.7 Mb genomic region, the smallest critical genomic region, for DS-associated heart defects, and our results should set the stage for the final step to establish the identities of the causal gene(s), whose elevated expression(s) directly underlie this major DS phenotype.
Assuntos
Cromossomos de Mamíferos , Síndrome de Down/genética , Genoma , Cardiopatias Congênitas/genética , Coração/embriologia , Animais , Mapeamento Cromossômico , Cromossomos Humanos Par 21 , Modelos Animais de Doenças , Síndrome de Down/embriologia , Síndrome de Down/patologia , Embrião de Mamíferos , Feminino , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/genética , Dosagem de Genes , Engenharia Genética , Loci Gênicos , Fatores de Troca do Nucleotídeo Guanina/genética , Cardiopatias Congênitas/embriologia , Cardiopatias Congênitas/patologia , Humanos , Masculino , Camundongos , Fenótipo , Recombinação Genética , Sintenia , Proteína 1 Indutora de Invasão e Metástase de Linfoma de Células TRESUMO
Human trisomy 21, the chromosomal basis of Down syndrome (DS), is the most common genetic cause of heart defects. Regions on human chromosome 21 (Hsa21) are syntenically conserved with three regions located on mouse chromosome 10 (Mmu10), Mmu16 and Mmu17. In this study, we have analyzed the impact of duplications of each syntenic region on cardiovascular development in mice and have found that only the duplication on Mmu16, i.e., Dp(16)1Yey, is associated with heart defects. Furthermore, we generated two novel mouse models carrying a 5.43-Mb duplication and a reciprocal deletion between Tiam1 and Kcnj6 using chromosome engineering, Dp(16Tiam1-Kcnj6)Yey/+ and Df(16Tiam1-Kcnj6)Yey/+, respectively, within the 22.9-Mb syntenic region on Mmu16. We found that Dp(16Tiam1-Kcnj6)Yey/+, but not Dp(16)1Yey/Df(16Tiam1-Kcnj6)Yey, resulted in heart defects, indicating that triplication of the Tiam1-Knj6 region is necessary and sufficient to cause DS-associated heart defects. Our transcriptional analysis of Dp(16Tiam1-Kcnj6)Yey/+ embryos confirmed elevated expression levels for the genes located in the Tiam-Kcnj6 region. Therefore, we established the smallest critical genomic region for DS-associated heart defects to lay the foundation for identifying the causative gene(s) for this phenotype.
Assuntos
Síndrome de Down/genética , Cardiopatias Congênitas/genética , Animais , Modelos Animais de Doenças , Feminino , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/genética , Duplicação Gênica/genética , Fatores de Troca do Nucleotídeo Guanina/genética , Masculino , Camundongos , Camundongos Mutantes , Deleção de Sequência/genética , Sintenia/genética , Proteína 1 Indutora de Invasão e Metástase de Linfoma de Células TRESUMO
TBX1 is thought to be a critical gene in the pathogenesis of del22q11/DiGeorge syndrome (DGS). Morphological abnormalities of the external ear and hearing impairment (conductive or sensorineural) affect the majority of patients. Here we show that homozygous mutation of the mouse homolog Tbx1 is associated with severe inner ear defects that prevent the formation of the cochlea and of the vestibulum. Consistent with phenotypic abnormalities, Tbx1 is expressed early in otocyst development in the otic epithelium and in the periotic mesenchyme. Tbx1 loss-of-function blocks inner ear development at early otocyst stage and after neurogenesis. Analysis of chimeras suggests that Tbx1 function is required in the otic epithelium cell autonomously, but abnormalities of the periotic mesenchyme indicate that the pathogenesis of the inner ear phenotype is complex. We propose a model where Tbx1 is required for expansion of a subpopulation of otic epithelial cells, which is required to form the vestibular and auditory organs. Our data suggest that Tbx1 deletion in del22q11 patients may cause not only external and middle ear defects but also sensorineural and vestibular phenotypes observed in these patients.