Perturbations of Vascular Homeostasis and Aortic Valve Abnormalities in Fibulin-4 Deficient Mice

Abstract

Abstract-The Fibulins are a 6-member protein family hypothesized to function as intermolecular bridges that stabilize the organization of extracellular matrix structures. Here, we show that reduced expression of Fibulin-4 leads to aneurysm formation, dissection of the aortic wall and cardiac abnormalities. Fibulin-4 knockdown mice with a hypomorphic expression allele arose from targeted disruption of the adjacent Mus81 endonuclease gene. Mice homozygous for the Fibulin-4 reduced expression allele (Fibulin-4 R/R ) show dilatation of the ascending aorta and a tortuous and stiffened aorta, resulting from disorganized elastic fiber networks. They display thickened aortic valvular leaflets that are associated with aortic valve stenosis and insufficiency. Strikingly, already a modest reduction in expression of Fibulin-4 in the heterozygous ϩ/R mice occasionally resulted in small aneurysm formation. To get insight into the underlying molecular pathways involved in aneurysm formation and response to aortic failure, we determined the aorta transcriptome of ϩ/R and Fibulin-4 R/R animals and identified distinct and overlapping biological processes that were significantly overrepresented including cytoskeleton organization, cell adhesion, apoptosis and several novel gene targets. Transcriptome and protein expression analysis implicated perturbation of TGF-␤ signaling in the pathogenesis of aneurysm in fibulin-4 deficient mice. Our results show that the dosage of a single gene can determine the severity of aneurysm formation and imply that disturbed TGF-␤ signaling underlies multiple aneurysm phenotypes. (Circ Res. 2007;100:738-746.)Key Words: aneurysm Ⅲ aortic valve Ⅲ mouse model E xtracellular elastic fibers supply structure and mechanical elasticity to organs such as large arteries, lungs and skin. 1 Elastic fibers are assembled through polymerization of tropoelastin monomers and loss of elastin is associated with aneurysmal degeneration of the aorta. 2 The 6-member protein family of Fibulins, which are prominently expressed in blood vessels are hypothesized to function as intermolecular bridges that stabilize the organization of extracellular structures such as elastic fibers and basement membranes. 3 Fibulin-4 is found in the medial layers of arteries and heart valves. 4 Whereas complete Fibulin-4 knockout mice have recently been generated 5 and showed embryonic lethality (E12.5), we generated a Fibulin-4 allele with reduced expression by transcriptional interference through placement of a TKneo targeting construct in a downstream gene (Mus81). Mus81 knockout mice have been generated previously. 6,7 In the targeting strategy of Dendouga et al, exons 9 to 12 were replaced by a PGKneo marker flanked by loxP sites and subsequently the marker was excised using Cre-recombinase expressing mice. Mus81 knockout mice from which the selectable marker was removed were born at expected Mendelian frequencies and were indistinguishable from WT littermates in terms of development, growth, immune function and fertility. 6 To examine ...

Assembly of fibrillin microfibrils governs extracellular deposition of latent TGFβ

“…Thus, in MAGP-1-null mice (Weinbaum et al, 2008), lack of competition for LLC binding to microfibrils could result in enhanced microfibril-bound LLC and reduced active TGF. By contrast, enhanced TGF activity in mice depleted of fibulin-4 (Hanada et al, 2007) suggests a stabilizing role for fibulin-4, which is consistent with our finding that, unlike MAGP-1, exogenous fibulin-4 does not release TGF from microfibrils. We have also shown that fibulin-4 can strongly bind LTBP-1, and can simultaneously bind LTBP-1 and fibrillin-1, implicating fibulin-4 as a key mediator in the association of LTBP-1 with microfibrils (Fig.…”

“…Fibulin-4 binds strongly to full-length LTBP-1 and fibrillin-1 Fibulin-4 binds fibrillin-1 (Choudhury et al, 2009;Ono et al, 2009) and colocalizes with microfibrils (Kobayashi et al, 2007), and fibulin-4 hypomorphic mice have enhanced TGF activity (Hanada et al, 2007). Here, BIAcore analysis revealed that fulllength LTBP-1 bound strongly to immobilized full-length fibulin-4 (K D 2.1±0.12 nM) (Fig.…”

“…The importance of microfibrils in TGF regulation is highlighted by pathologically elevated TGF activity in the connective-tissue disorder Marfan syndrome, which is caused by fibrillin-1 mutations that disrupt the formation and/or stability of tissues such as blood vessels, eyes and bones (Robinson et al, 2006). Mice depleted of fibulin-4, a microfibril-associated molecule, have elastic fibre defects (McLaughlin et al, 2006;Hanada et al, 2007;Horiguchi et al, 2009) and exhibit enhanced aortic TGF activity (Hanada et al, 2007). By contrast, mice lacking microfibril-associated glycoprotein MAGP-1 (also known as MFAP-2) have reduced TGF activity but no overt microfibril defects (Weinbaum et al, 2008).…”

Periostin advances atherosclerotic and rheumatic cardiac valve degeneration by inducing angiogenesis and MMP production in humans and rodents

“…Although the onset of aortic valve stenosis has been reported in Smad6-deficient mice (14), in fibulin 4-deficient mice (15), and in humans with the NOTCH1 mutation (16), the abnormality has primarily been observed during cardiac valve development. As avascular tissues, the cardiac valve complex and cartilage share common structural properties (17,18).…”

MicroRNAs, fibrotic remodeling, and aortic aneurysms

“…Using a mouse model of thoracic aortic disease, the FBLN4 hypomorphic mouse, they found that miR-29a, miR-29b, and miR-29c were increased in aortic aneurysms in the mutant mouse. Aortic disease in the FBLN4 mouse model is associated with evidence of increased TGF-b signaling, including increased nuclear phosphorylated Smad2 (pSmad2) and increased connective tissue growth factor (CTGF) and collagen deposition in the medial and adventitial layer (22). Therefore, it is surprising that miR-29b would be increased rather than decreased in the diseased aorta.…”