Understanding how defects in familial thoracic aneurysm and dissection (FTAAD)‐associated mutations in MAGP2 (MFAP5) affect its extracellular matrix localization and regulation of TGF‐β/SMAD signaling
Authors: Miyamoto Alison , Daniels Ryan , Burcham Austin , Keith Amber , Luthra Manpreet 
Published: April 2020

In familial thoracic aneurysm and dissection (FTAAD), the aorta becomes weakened, stretching the vessel, a process known as aortic dilation, which in extreme cases can lead to tearing of the vessel wall layers, commonly referred to as aortic dissection. The direct consequences of FTAAD are stroke or heart attack that can potentially result in death of the patient. Research on Marfan Syndrome (MFS) and Loeys‐Dietz Syndrome (LDS) have shown that genes associated with similar aortic abnormalities have alterations in the TGF‐β/SMAD signaling pathway as well as extracellular matrix proteins (Faivre et al., 2007, Mizuguchi et al., 2004, and Loeys et al., 2006). Mutations in MFAP5, a gene that encodes for microfibril‐associated glycoprotein 2 (MAGP2), have been found in two FTAAD families, a nonsense mutation R158* and a missense mutation W21L (Barbier et al., 2014). The current working hypothesis is that MAGP2 normally plays an inhibitory role in TGF‐β/SMAD signaling, and that the FTAAD‐associated mutations are loss of function, since aortic tissue from a patient with the W21L mutation showed high phospho‐SMAD (p‐SMAD) levels. Moreover, MAGP2 has been shown to directly bind to active TGF‐β1 in vitro (Combs et al., 2013), and we have confirmed the results of Barbier et al. that the R158* mutant is poorly secreted. In contrast, the defect in the W21L mutant protein remains unidentified. It was secreted and bound to extracellular fibrillin microfibrils similar to wild‐type MAGP2 in our cell culture studies. What remains untested is whether FTAAD‐associated MAGP2 mutant proteins directly or indirectly affect TGF‐β/SMAD signaling. Therefore, the objective of this study is to determine whether R158* MAGP2 and W21L MAGP2 mutant proteins lose the ability to regulate TGF‐β signaling, leading to an increase in p‐SMAD2/3 levels in the cell. A TGF‐β signaling assay will be used to test soluble MAGP2 proteins, and fluorescent western blotting will then quantify the level of p‐SMAD 2/3 in whole cell lysate of MG63 cells, which expresses the TGF‐β receptor. Verification of both p‐SMAD2/3 and total SMAD antibodies have been completed via standard chemiluminescent protocols, and are currently being optimized for fluorescent detection. In parallel, stable cell lines are being produced to express wild‐type, R158*, or W21L MAGP2. A cell line expressing wild‐type MAGP2 has been generated, and the other two cell lines are being checked via immunofluorescence and western blotting to confirm that the cell lines express MAGP2. An understanding of how MAGP2 regulates the TGF‐β signaling could lead to the engineering of drugs to target mutant MAGP2 in hopes of improving the outcomes of FTAAD.Support or Funding InformationThis work was supported by the Department of Biological Science research funds (A. Miyamoto), CIRM Bridges to Stem Cell Research (A. Burcham, A. Keith), and HHMI (R. Daniels).