Fibrillins 1 and 2 perform partially overlapping functions during aortic development

Abstract

Fibrillin-rich microfibrils are extracellular assemblies that impart structural properties to the connective tissue. To elucidate the contribution of fibrillin-rich microfibrils to organogenesis, we have examined the vascular phenotype of a newly created strain of mice that completely lacks fibrillin-1 and the consequences of combined deficiency of fibrillins 1 and 2 on tissue formation. The results demonstrated that fibrillins 1 and 2 perform partially overlapping functions during aortic development. Fbn1-/- mice died soon after birth from ruptured aortic aneurysm, impaired pulmonary function, and/or diaphragmatic collapse. Analysis of the neonatal Fbn1-/- aorta documented a disorganized and poorly developed medial layer but normal levels of elastin cross-links. Transcriptional profiling revealed that aneurysm progression in Fbn1 null mice is accompanied by unproductive up-regulation of gene products normally involved in tissue repair and vascular integrity, such as plasminogen activator inhibitor-1, activin A, and cysteine-rich angiogenic protein 61. In contrast to Fbn1-/- mice, Fbn2 null mice had a well developed and morphologically normal aortic wall. However, virtually all Fbn1-/-;Fbn2-/- embryos and about half of the Fbn1+/-;Fbn2-/- embryos died in utero and displayed a significantly more severe vascular phenotype than Fbn1-/- mice. Consistent with a specialized function of fibrillin-2, electron microscopy visualized ultrastructurally different microfibrils in Fbn1 null compared with control cell cultures. Collectively, these data demonstrate that involvement of fibrillin-2 in the initial assembly of the aortic matrix overlaps in part with fibrillin-1 and that continued fibrillin-1 deposition is absolutely required for the maturation and function of the vessel during neonatal life.

FIGURE 1. Fbn1 gene targeting strategy

A, top to bottom, the wild-type genomic region that was targeted with the size of the wild-type EcoRI restriction fragment detected by probe A in the Southern analysis, the targeting vector containing the human alkaline phosphatase (hAP) and the phosphoglycerate kinase -neo (Neo) selectable gene with the arrow indicating the direction of transcription, and the targeted mgN allele with the size of the mutant EcoRI fragment detected by probe A. IRES, internal ribosome entry site. kb, kilo-bases. B, Southern blots of tail DNA from wild-type (+/+), heterozygous (+/−), and homozygous (−/−) mgN mice that was digested with EcoRI and hybridized to probe A; the size of DNA fragments (in kilobases) is indicated on the left side of the autoradiogram. C, Northern blots of RNA from lung tissues of wild-type (+/+) and mgN/mgN (−/−) mice hybridized to Fbn1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probes. D, Western blots of conditioned media from wild-type (+/+) and mgN/mgN (−/−) mouse embryonic fibroblast cultures using anti-fibrillin-1 or fibrillin-2 antibodies (α); the size of fibrillins (in kDa) is indicated on the left side of the autoradiogram.

FIGURE 2. Fbn1 null phenotype

A, left two panels, representative aneurysm of the ascending aorta in a newborn mouse with histological documentation (Weigert staining) of elastic fiber fragmentation and aortic wall rupture (arrow). Right four panels, top, right lungs of wild-type (+/+) and Fbn1 null (−/−) mice showing multiple blebs in the peripheral portion of the latter (arrow); bottom, partial views of the exposed thoraxes of wild-type and mutant mice showing thinner intercostal muscles and abnormal ribs in the latter compared with the former animal. B, cross-sections of aortic tissue around the lesions in mutant mice (−/−) and at corresponding levels in age and sex-matched wild-type littermates stained with H&E, Weigert, trichrome, or anti-MMP-9 antiserum. On the right side of the H&E panels is a magnification of the mutant tissue showing an altered architecture with loss of normal contacts between cells and with the surrounding matrix (yellow arrows). The immunoblot on the right further documents elevated MMP-9 compared with β-tubulin levels in the decaying post-mortem aorta of mutant mice. All the specimens are oriented with the lumen of the vessel at the top; scale bar = 100 μm.

FIGURE 3. Histopathology of Fbn1 null aortas

A, immunostaining with anti-Von Willebrand factor antibody of cross-sections of aortas from a randomly sacrificed (P14) and a dead (pm) Fbn1 null (−/−) mouse showing detachment of the endothelial lining (arrows). Control samples were from sex- and age-matched wild-type (+/+) littermates. The signal was pseudocolored using Adobe Photoshop software to best appreciate the immunostaining. B, Weigert staining of aortic tissues in segments other than the focal lesions of Fbn1 null mice (−/−) and in comparable regions of the wild-type (+/+) aorta. Arrows indicate discontinuities in elastic fibers. Scale bar, 100 μm in both panels.

FIGURE 4. Aortic development in Fbn1 null mice

A, cross-sections of aortas from wild-type (+/+) and mgN/mgN (−/−) embryos (E16.5), and randomly sacrificed newborn mice (P1, P10, and P14) were stained with H&E or Weigert for elastic fibers. B, aortas from E16.5 and P14 wild-type (+/+) and mgN/mgN mice immunostained for SMemb, an embryonic VSMC marker. Scale bar, 50 μm in both panels.

FIGURE 5. Altered gene expression in Fbn1 null aorta

A, left, bar graphs indicate the mean -fold levels ± S.E. of Pai-1, Cyr61/Ccn1, and activin A transcripts in the aortas of P14 wild-type (open bars) and mutant (filled bars) mice (n = 3 per genotype) as evaluated by real-time PCR. The asterisk indicates statistically significant differences between samples. Right, illustrative immunostaining of PAI-1 and CYR61/CCN1 in the aortas of P14 wild-type (+/+) and mutant (−/−) mice; scale bar, 100 μm. B, left, Western analysis of phosphorylated Smad2 and β-tubulin protein levels in the aortas of P14 wild-type (+/+) and mutant (−/−) mice. Right, bar graphs summarizing the data upon normalization of the Western blot experiments. Bars show the means ± S.E., and the asterisk indicates statistically significant differences between control (open bars) and mutant (filled bars) sample (n = 3 per genotype). β-tub, β-tubulin.

FIGURE 6. Elastic fibers in the aorta of Fbn2 null mice

Weigert-stained cross-sections of ascending aortas from wild-type (WT) (+/+) and Fbn2 null (−/−) embryos (E14.5) and randomly sacrificed P1 and P30 mice. Scale bars, 100 μm in the top four panels and 20 μm in the bottom two.

FIGURE 7. Phenotype of double fibrillin mutant embryos

Top, illustrative examples of wild-type (WT) and Fbn1^−/−;Fbn2 ^−/− embryos at E14.5. Bottom, Weigert-stained cross-sections of ascending aortas from the embryos shown above as well as from an additional double null embryo and a Fbn1 ^+/−;Fbn2 ^−/− embryo documenting poor organization of the vessel wall in the mutant samples. Scale bar, 50 μm.

FIGURE 8. Ultrastructural morphology of fibrillin polymers

Microfibrils purified from wild-type (A), mgN/+ (C), or mgN/mgN (B and D) neonatal fibroblast cultures are compared after rotary shadowing and electron microscopy. Wild-type and mgN/+ microfibrils contain the normal and half the normal amount of fibrillin-1, respectively, and significantly less fibrillin-2. Instead, mgN/mgN microfibrils are composed exclusively of fibrillin-2. A density just to one side of each globular domain was seen only in the fibrillin-2 microfibrils and best visualized after negative staining of the fibrillin-2 microfibrils (D). Similar electron densities were occasionally observed in microfibrils from mgN/+ fibroblast cultures (C) Arrows point to the globular beads, and arrowheads mark some of the densities. Scale bar, 100 nm.