Characterization of the skeletal phenotype of the mg?loxPneo mouse model of Marfan syndrome and analysis of the mechanisms of pathogenesis

Author(s)

Elisa Ito Kawahara

• Document type: Master's Dissertation
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Instituto de Biociências (IBIOC/SB)
• Defense date: 2016-12-02

Examining board members

Lygia da Veiga Pereira Carramaschi; Cecilia Helena de Azevedo Gouveia Ferreira; Ana Beatriz Alvarez Perez

• Advisor: Lygia da Veiga Pereira Carramaschi; Luis Ernesto Farinha Arcieri

Abstract

Marfan syndrome (MFS) is an autosomal dominant disease that affects the connective tissue. The main clinical manifestations affect the cardiovascular, optical and bone systems. MFS is caused by mutations in the FBN1 gene, that encodes the extracellular protein fibrillin-1, a major component of microfibrils, which form elastic fibers. Studies have shown that mutations in the fibrillin-1 gene lead to an indiscriminate increase in active TGF-Β in the matrix, which results in the major phenotypes of the disease. The Renin Angiotensin System (RAS) has as its main product Angiotensin II (Ang-II), involved in bone mass regulation and TGF-Β activity. Therapeutic strategies using drugs targeting the RAS have been studied in animal models. Ramipril, an ACE inhibitor (Angiotensin-converting enzyme inhibitor, ACEi), increase Fbn1 gene expression in 35% and improve kyphosis index in the mgΔloxPneo mouse model for SMF. Its mechanism of action in bone tissue is not completely elucidated, and it may act by decreasing Ang-II production and consequent reduction in TGF-Β levels, or by inhibiting degradation of bradykinin (BK) by Ang II. BK directly activates its B2R receptor, which induces opposite physiological actions to Ang-II. This study aims to evaluate and understand the general mechanisms of bone pathogenesis in the mgΔloxPneo mouse model. We analyzed the bone phenotype of mgΔloxPneo and wildtype animals treated, or not, with Ramipril by measuring the kyphosis index (KI), micro computed tomography (μCT) and Real-time PCR (RT-PCR). We found that mutant animals showed a greater degree of kyphosis and an altered bone structure. Ramipril improved kyphosis but did not alter bone quality of mutant animals, while in wild type animals Ramipril decreased bone structure without altering KI. Therefore, the beneficial effect of Ramipril on mgΔloxPneo animals\' kyphosis is not due to an improvement in bone structure. In order to test the hypothesis where signaling through BK B2R receptor may be involved in the development of bone phenotype of mgΔloxPneo animals, a mouse model with the mgΔloxPneo mutation and knockout for B2R receptor was generated. The analysis of these animals show that the B2R receptor does not interfere with the development of kyphosis, with Fbn1 genotype as sole determinant for this phenotype manifestation. RNA-seq analysis was performed to verify differential expression of genes and altered cellular pathways, which could reveal mechanisms of bone phenotype development in mgΔloxPneo animals. Altered pathways found included focal adhesion, receptor- extracellular matrix (ECM) interaction, tight junction, nucleotide excision repair and missmatch repair, which may explain changes in bone cells metabolism. In addition, there were differences in gene expression related to skeletal muscle metabolism, which is in agreement with the paracrine regulation of bone and muscle tissue, leading to worst bone structure (AU)

• FAPESP's process: 13/20363-0 - Participation B2R bradykinin receptor in phenotypic improvement induced by Ramipril in a murine model for Marfan Syndrome
• Grantee: Elisa Ito Kawahara
• Support Opportunities: Scholarships in Brazil - Master