RESUMO
RATIONALE: Endothelial cells have the ability to undergo endothelial-mesenchymal transitions (EndMTs), by which they acquire a mesenchymal phenotype and stem cell-like characteristics. We previously found that EndMTs occurred in the endothelium deficient in matrix γ-carboxyglutamic acid protein enabling endothelial cells to contribute cells to vascular calcification. However, the mechanism responsible for initiating EndMTs is not fully understood. OBJECTIVE: To determine the role of specific serine proteases and sex determining region Y-box 2 (Sox2) in the initiation of EndMTs. METHODS AND RESULTS: In this study, we used in vivo and in vitro models of vascular calcification to demonstrate that serine proteases and Sox2 are essential for the initiation of EndMTs in matrix γ-carboxyglutamic acid protein-deficient endothelium. We showed that expression of a group of specific serine proteases was highly induced in endothelial cells at sites of vascular calcification in Mgp null aortas. Treatment with serine protease inhibitors decreased both stem cell marker expression and vascular calcification. In human aortic endothelial cells, this group of serine proteases also induced EndMTs, and the activation of proteases was mediated by Sox2. Knockdown of the serine proteases or Sox2 diminished EndMTs and calcification. Endothelial-specific deletion of Sox2 decreased expression of stem cell markers and aortic calcification in matrix γ-carboxyglutamic acid protein-deficient mice. CONCLUSIONS: Our results suggest that Sox2-mediated activation of specific serine proteases is essential for initiating EndMTs, and thus, may provide new therapeutic targets for treating vascular calcification.
Assuntos
Calcinose , Endotélio Vascular/metabolismo , Mesoderma/metabolismo , Serina Endopeptidases/metabolismo , Animais , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Células Cultivadas , Endotélio Vascular/patologia , Endotélio Vascular/ultraestrutura , Ativação Enzimática , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Perfilação da Expressão Gênica/métodos , Humanos , Immunoblotting , Calicreínas/genética , Calicreínas/metabolismo , Mesoderma/patologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Elastase Pancreática/genética , Elastase Pancreática/metabolismo , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Serina Endopeptidases/genética , Proteína 1 Relacionada a Twist/genética , Proteína 1 Relacionada a Twist/metabolismo , Proteína de Matriz GlaRESUMO
Diabetes mellitus affects the adipose tissue and mesenchymal stem cells derived from the adipose stroma and other tissues. Previous reports suggest that bone morphogenetic protein 4 (BMP4) is involved in diabetic complications, at the same time playing an important role in the maintenance of stem cells. In this study, we used rats transgenic for human islet amyloid polypeptide (HIP rats), a model of type 2 diabetes, to study the effect of diabetes on adipocyte-derived stem cells, referred to as dedifferentiated fat (DFAT) cells. Our results show that BMP4 expression in inguinal adipose tissue is significantly increased in HIP rats compared to controls, whereas matrix Gla protein (MGP), an inhibitor of BMP4 is decreased as determined by quantitative PCR, and immunofluorescence. In addition, adipose vascularity and expression of multiple endothelial cell markers was increased in the diabetic tissue, visualized by immunofluorescence for endothelial markers. The endothelial markers co-localized with the enhanced BMP4 expression, suggesting that vascular cells play a role BMP4 induction. The DFAT cells are multipotent stem cells derived from white mature adipocytes that undergo endothelial and adipogenic differentiation. DFAT cells prepared from the inguinal adipose tissue in HIP rats exhibited enhanced proliferative capacity compared to wild type. In addition, their ability to undergo both endothelial cell and adipogenic lineage differentiation was enhanced, as well as their response to BMP4, as assessed by lineage marker expression. We conclude that the DFAT cells are affected by diabetic changes and may contribute to the adipose dysfunction in diabetes.
Assuntos
Proteína Morfogenética Óssea 4/biossíntese , Diferenciação Celular/genética , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Tipo 2/genética , Tecido Adiposo , Animais , Animais Geneticamente Modificados , Proteína Morfogenética Óssea 4/genética , Proteínas de Ligação ao Cálcio/biossíntese , Proteínas de Ligação ao Cálcio/genética , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 2/patologia , Proteínas da Matriz Extracelular/biossíntese , Proteínas da Matriz Extracelular/genética , Humanos , Polipeptídeo Amiloide das Ilhotas Pancreáticas/biossíntese , Polipeptídeo Amiloide das Ilhotas Pancreáticas/genética , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/patologia , Ratos , Proteína de Matriz GlaAssuntos
Proteínas de Ligação ao Cálcio/genética , Proteínas da Matriz Extracelular/genética , Pneumopatias , Telangiectasia Hemorrágica Hereditária/genética , Malformações Vasculares/genética , Receptores de Ativinas Tipo I/genética , Receptores de Activinas Tipo II , Animais , Proteína Morfogenética Óssea 4/genética , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Humanos , Pneumopatias/etiologia , Pneumopatias/genética , Camundongos , Modelos Genéticos , Circulação Pulmonar/genética , Proteína de Matriz GlaRESUMO
An 18-year-old virginal woman was referred to the reproductive endocrinology clinic with primary amenorrhoea and secondary sexual development in the absence of pelvic pain. Additionally, she had significant congenital sensorineural hearing loss, autism, bipolar disorder and class III obesity. On physical examination, secondary sexual development was confirmed (Tanner 5 breasts and Tanner 4 pubic hair). She refused further pelvic examination following prior attempts by the referring physicians. Serum leutinizing hormone (LH), follicle sitmulating hormone (FSH). prolactin, estradiol and total testosterone values were within normal limits. Karyotype was 46,XX. MRI demonstrated complete uterine agenesis, short vagina, sacral dysgenesis with complete absence of the coccyx and a horseshoe kidney. Diagnosis of Mayer-Rokitansky-Küster-Hauser Syndrome type 2 was established based on clinical, laboratory and MRI findings. The patient and family were counselled regarding the disease process, techniques for vaginal elongation, sexual activity and future reproductive options.