Altered TGFbeta signaling and cardiovascular manifestations in patients with autosomal recessive cutis laxa type I caused by fibulin-4 deficiency

Abstract

Fibulin-4 is a member of the fibulin family, a group of extracellular matrix proteins prominently expressed in medial layers of large veins and arteries. Involvement of the FBLN4 gene in cardiovascular pathology was shown in a murine model and in three patients affected with cutis laxa in association with systemic involvement. To elucidate the contribution of FBLN4 in human disease, we investigated two cohorts of patients. Direct sequencing of 17 patients with cutis laxa revealed no FBLN4 mutations. In a second group of 22 patients presenting with arterial tortuosity, stenosis and aneurysms, FBLN4 mutations were identified in three patients, two homozygous missense mutations (p.Glu126Lys and p.Ala397Thr) and compound heterozygosity for missense mutation p.Glu126Val and frameshift mutation c.577delC. Immunoblotting analysis showed a decreased amount of fibulin-4 protein in the fibroblast culture media of two patients, a finding sustained by diminished fibulin-4 in the extracellular matrix of the aortic wall on immunohistochemistry. pSmad2 and CTGF immunostaining of aortic and lung tissue revealed an increase in transforming growth factor (TGF)beta signaling. This was confirmed by pSmad2 immunoblotting of fibroblast cultures. In conclusion, patients with recessive FBLN4 mutations are predominantly characterized by aortic aneurysms, arterial tortuosity and stenosis. This confirms the important role of fibulin-4 in vascular elastic fiber assembly. Furthermore, we provide the first evidence for the involvement of altered TGFbeta signaling in the pathogenesis of FBLN4 mutations in humans.

Figure 1

(a) Clinical photograph of patient 3 showing a prominent forehead with depressed nasal bridge, mild hypertelorism and downslanting palpebral fissures. The patient has a trachea canulae. (b) MRI shows severe tortuosity of the carotid arteries (anterolateral oblique view). (c) A posterior view of a 3D reconstruction of an MR angio showing, successively, the dilated aorta ascendens (indicated with an arrow), hypoplasia of the transverse aorta (indicated with an arrowhead) and tortuous descending aorta.

Figure 2

DNA sequence of FBLN4 in (a) patient 1, showing the missense c.376G>A mutation, leading to Glu126Lys substitution in the protein. The glutamic acid residue at position 126 is evolutionarily conserved and located in the DINE consensus sequence of the second cbEGF-like domain, showing in (b) patient 2 the c.1189G>A mutation, leading to the Ala397Thr substitution. The alanine residue is located in the C-terminal fibulin-like module and is conserved between fibulin homologs and highly conserved among species, showing in (c) patient 3 the compound heterozygous c.377A>T missense (leading to the Glu126Val substitution) and c.577delC mutations. The missense mutation affects the same conserved glutamic acid residue as identified in patient 1 (see panel a for alignment of the conserved sequence).

Figure 3

(a) Immunohistochemical staining of aortic tissue of patient 4 and its age- and sex-matched control. Intracellular fibulin-4 staining is seen in both individuals (examples of intracellular staining are indicated with a black arrow). No extracellular fibulin-4 is seen in the aorta of the patient, compared with its control (extracellular staining is indicated with white arrows). Scale bars=100 μm. (b) Immunoblot analysis of cultured media using fibroblasts of age- and sex-matched controls (C1 and C5) and of two patients (P1 and P5). Top panel: anti-fibulin-4 monoclonal antibody (clone 347) demonstrating impaired fibulin-4 secretion in patient 5 and slightly impaired fibulin-4 secretion in patient 1 in contrast to their matched control. Bottom panel: anti-fibronectin-1 monoclonal antibody indicating the amounts of samples loaded on the gel. The fibulin-4 secretion to the extracellular space as seen on the gel images was quantified using ImageJ software.

Figure 4

Immunohistochemical staining of aorta (a–d and i–l) and lung (e–h and m–p) tissue in FBLN4 mutation-positive patient 4 (b, d, f, h, j, l, n, p) and in control (a, c, e, g, i, k, m, o). Increased nuclear accumulation of phosphorylated Smad2 is present in both the aorta (b ,d) (some of the nuclei are indicated with an arrow) and the lung (f, h) of the patient, as compared with control aorta (a, c) and lung (e, g). Similarly, increased levels of CTGF expression are noted in the aorta (j, l) and lung (n, p) of the patient, compared with control aorta (i, k) and lung (m, o). These results are indicative of increased TGFβ signaling in the FBLN4 mutation-positive patient. Scale bars=100 μm.

Figure 5

Immunoblot analysis of fibroblast cell extracts of age- and sex-matched controls (C1 and C5) and patients 1 (P1) and 5 (P5). The graph and the pSmad2 immunoblotting panel shows an increase in pSmad2 level in patient 1 and patient 5 when cells were stimulated with TGFβ protein, in contrast to their controls. When cells are not stimulated, basal expression of pSmad2 is seen. Bottom panel: anti-vinculin antibody, indicating the amounts of samples loaded on the gel. This immunoblotting analysis is representative of three independent experiments (see Supplementary Figure S1).