Function of Ltbp-4L and fibulin-4 in survival and elastogenesis in mice

ABSTRACT LTBP-4L and LTBP-4S are two isoforms of the extracellular matrix protein latent-transforming growth factor beta-binding protein 4 (LTBP-4). The mutational inactivation of both isoforms causes autosomal recessive cutis laxa type 1C (ARCL1C) in humans and an ARCL1C-like phenotype in Ltbp4−/− mice, both characterized by high postnatal mortality and severely affected elastogenesis. However, genetic data in mice suggest isoform-specific functions for Ltbp-4 because Ltbp4S−/− mice, solely expressing Ltbp-4L, survive to adulthood. This clearly suggests a requirement of Ltbp-4L for postnatal survival. A major difference between Ltbp4S−/− and Ltbp4−/− mice is the matrix incorporation of fibulin-4 (a key factor for elastogenesis; encoded by the Efemp2 gene), which is normal in Ltbp4S−/− mice, whereas it is defective in Ltbp4−/− mice, suggesting that the presence of Ltbp-4L might be required for this process. To investigate the existence of a functional interaction between Ltbp-4L and fibulin-4, we studied the consequences of fibulin-4 deficiency in mice only expressing Ltbp-4L. Resulting Ltbp4S−/−;Fibulin-4R/R mice showed a dramatically reduced lifespan compared to Ltbp4S−/− or Fibulin-4R/R mice, which survive to adulthood. This dramatic reduction in survival of Ltbp4S−/−;Fibulin-4R/R mice correlates with severely impaired elastogenesis resulting in defective alveolar septation and distal airspace enlargement in lung, and increased aortic wall thickness with severely fragmented elastic lamellae. Additionally, Ltbp4S−/−;Fibulin-4R/R mice suffer from aortic aneurysm formation combined with aortic tortuosity, in contrast to Ltbp4S−/− or Fibulin-4R/R mice. Together, in accordance with our previous biochemical findings of a physical interaction between Ltbp-4L and fibulin-4, these novel in vivo data clearly establish a functional link between Ltbp-4L and fibulin-4 as a crucial molecular requirement for survival and elastogenesis in mice. Summary: The interaction of the long form of latent-transforming growth factor beta-binding protein 4 and fibulin-4 is essential for survival as well as for the formation of elastic fibers.


INTRODUCTION
Latent-transforming growth factor beta-binding protein 4 (LTBP-4) is expressed, secreted and deposited within the extracellular matrix (ECM) as a short (LTBP-4S) and a long (LTBP-4L) isoform . In humans and mice, both isoforms show distinct tissue expression patterns in various organs (Bultmann-Mellin et al., 2015;Kantola et al., 2010), which has been proposed to result from the activation of two known LTBP-4 promoters under the control of independent transcription factors (Bultmann et al., 2013;Kantola et al., 2010).
In humans, mutations in the LTBP4 gene cause functional modifications of both LTBP-4 isoforms, leading to autosomal recessive cutis laxa type 1C (ARCL1C; initially called Urban-Rifkin-Davis syndrome). ARCL1C is a rare congenital connective tissue disorder characterized by high mortality in the first months of life due to impaired elastic fiber architecture in several visceral organs, including massive emphysemas in the lung leading to early death by respiratory failure (Callewaert et al., 2013;Urban et al., 2009).
In lungs from all analyzed genotypes, Ltbp-4 and fibulin-4 immunoreactivity was found in the bronchial and bronchiolar walls, in the pulmonary parenchyma and vascular walls (Fig. S1). Lungs from Ltbp4S −/− , Fibulin-4 R/R and Ltbp4S −/− ;Fibulin-4 R/R mice showed enlarged alveolar spaces with reduced numbers of alveoli compared to WT lungs (Fig. 1D). However, lungs of Ltbp4S −/− ; Fibulin-4 R/R mice showed a higher degree of severely enlarged alveolar spaces with multifocal areas of atelectasis and a lack of alveolar and lobular architecture compared to all other genotypes (Fig. 1D).
In aortas of all genotypes, Ltbp-4 immunoreactivity was present throughout the entire aortic wall, especially in the vicinity of the aortic elastic lamellae. Fibulin-4 immunoreactivity was also found throughout the entire aortic wall, but was predominantly evident in the vicinity of the adventitia in aortas of all genotypes, and was strongly reduced in Fibulin-4 R/R and Ltbp4S −/− ;Fibulin-4 R/R mice (Fig. S1). Ltbp4S −/− , Fibulin-4 R/R and Ltbp4S −/− ;Fibulin-4 R/R mice displayed malformed and tortuous aortas with dilatation of the lumen of the abdominal aortas (Fig. 1E). Aortic walls of Ltbp4S −/− ; Fibulin-4 R/R mice were significantly thicker compared to those of WT mice. Aortic walls of Ltbp4S −/− ;Fibulin-4 R/R mice showed a markedly increased thickness compared to Ltbp4S −/− and Fibulin-4 R/R mice (Fig. 1F,G) and revealed focally extensive intramural hemorrhages and destruction of the aortic wall with necrotic cellular debris, which can be associated with lesions of aortic aneurysms (Fig. 1H). These pathological changes were not observed in the aortas of WT, Ltbp4S −/− or Fibulin-4 R/R mice at P4 (Fig. 1F).

DISCUSSION
In this study, we investigated whether a functional interdependence between Ltbp-4L and fibulin-4 influences survival and phenotype of mice. Therefore, Ltbp4S −/− ;Fibulin-4 R/R mice were generated, which only express the Ltbp-4L isoform and show reduced fibulin-4 expression.
Both Ltbp4S −/− and Fibulin-4 R/R mice show only mild emphysema in the early postnatal period and develop severe emphysema later in life (Bultmann-Mellin et al., 2015;Ramnath et al., 2014;Sterner-Kock et al., 2002). Ltbp4S −/− ;Fibulin-4 R/R lungs showed already severe emphysema at P4, indicating that the mutual presence of both proteins (Ltbp-4L and fibulin-4) is essential for normal lung development. It has been described that fibrillin-1deficient (Fbn1 mgΔ/mgΔ ) mice display impaired distal alveolar Differences between groups were analyzed by two-way ANOVA, followed by Bonferroni correction. septation in the early postnatal period and develop emphysema at an older age (Neptune et al., 2003). In lungs of Fbn1 mgΔ/mgΔ mice, deposition and localization of elastin is normal. Additionally, increased activation of TGFβ signaling is found to be causative for the lung phenotype because it could be attenuated by perinatal application of a TGFβ-neutralizing antibody (Neptune et al., 2003). However, we found normal matrix deposition of fibrillin-1 (Fig. S4) with no alterations in expression levels of TGFβ downstream targets (Ctgf and Pai1; Fig. S5) in lungs of Ltbp4S −/− , Fibulin-4 R/R and Ltbp4S −/− ;Fibulin-4 R/R mice at P4. These data suggest that altered fibrillin-1 fiber structure or matrix deposition did not contribute to the development of emphysema in Ltbp4S −/− , Fibulin-4 R/R or Ltbp4S −/− ;Fibulin-4 R/R mice.
In humans or mouse models with arterial tortuosity syndrome (Cheng et al., 2009;Coucke et al., 2006), Loeys-Dietz syndrome (Gallo et al., 2014;Loeys et al., 2006) , aortic or arterial tortuosity is a commonly described feature. Increased aortic tortuosity is associated with a poorer prognosis in aortic diseases (Franken et al., 2015;Hatakeyama et al., 2001;Morris et al., 2011;Shirali et al., 2013), but the detailed mechanisms leading to tortuosity are still unknown (Morris, 2015). However, elastic fibers are altered in most cases of aortic tortuosity (Morris, 2015). Our finding of a functional interaction between Ltbp-4L and fibulin-4 might allow further insight into the pathogenesis for this condition to be gained.
TGFβ signaling is upregulated in aortas of adult Fibulin-4 R/R mice and its postnatal inhibition with angiotensin (Ang)II type 1 (AT1) receptor antagonist losartan improves lifespan of Fibulin-4 R/R mice, but does not affect aortic vessel wall structure. Prenatal treatment with losartan prevents elastic fiber fragmentation in the aortic media of Fibulin-4 R/R mice, indicating that altered TGFβ signaling is associated with disturbed elastic fiber structure (Moltzer et al., 2011). However, mRNA expression of TGFβ downstream targets (Ctgf and Pai1) showed no differences in aortas from WT, Ltbp4S −/− , Fibulin-4 R/R and Ltbp4S −/− ;Fibulin-4 R/R mice at P4 (Fig. S5), but this might be different in other age groups.
Analysis of the aortae of all genotypes revealed that disrupted aortic elastic lamellae appeared to be thicker than continuous lamellae. Fibulin-4 R/R mice showed only occasional disruptions and no thickening of the elastic lamellae compared to WT mice. In contrast, the aortic elastic lamellae were significantly disrupted and significantly thicker in Ltbp4S −/− mice compared to WT. A possible explanation for the described thickening of the elastic lamellae might be the presence of non-linearized tropoelastin, which spontaneously aggregates to form large globular structures in vitro (Tu and Weiss, 2010).
Ltbp4S −/− ;Fibulin-4 R/R mice displayed aortic wall thickening compared to WT mice. It is very likely that deposition of amorphous, proteinaceous material within the aortic wall was the cause for the increased aortic thickness, a feature that has already been described in Ltbp4 −/− (Bultmann-Mellin et al., 2015) and Fibulin-4 R/R (Hanada et al., 2007;Moltzer et al., 2011) mice.
Ltbp4S −/− ;Fibulin-4 R/R mice showed an increased mortality related to handling at P3-P4. These mice displayed intramural aortic hemorrhages and destruction of the aortic wall with intramural necrotic cellular debris (Fig. 1H). Aortic aneurysm formation and dissection is also described for adult Fibulin-4 R/R mice (Hanada et al., 2007). However, studies in humans also imply a role for LTBP-4 in this condition. Data from four geographically distinct case control studies showed that sizes and growth rate of abdominal aortic aneurysm significantly correlate with the presence of a particular LTBP4 single-nucleotide polymorphism (LTBP4 21011A>T genotype). These data indicate a possible contribution of LTBP-4 to abdominal aortic aneurysm progression (Thompson et al., 2010). Therefore, ablation of Ltbp-4L in a Fibulin-4 R/R background might further promote aneurysm formation and dissection in Ltbp4S −/− ;Fibulin-4 R/R mice.
Fibulin-4 expression is not only essential for elastogenesis but also for intact collagen fiber assembly and homeostasis. Mice with smooth-muscle-specific loss of fibulin-4 expression show altered fibrillar collagen localization with larger, poorly organized fibrils in aortic walls (Papke et al., 2015) and fibulin-4 knockout mice show upregulation of the neutrophil collagenase matrix metalloprotease-8 in aortic walls (Kim et al., 2015). It is possible that impaired collagen assembly or homeostasis might contribute to the aortic phenotype in Ltbp4S −/− ;Fibulin-4 R/R mice, which is under current investigation.
Mutations in the human EFEMP2 gene lead to ARCL1B, whereas mutations in the human LTBP4 gene are causative for ARCL1C (Callewaert and Urban, 2016;Loeys et al., 2015). There are several clinical similarities between ARCL1B and ARCL1C patients, such as cutis laxa, and craniofacial and pulmonary phenotypes, indicating a functional relationship between both genes. However, abnormal gastrointestinal and urinary development is only present in individuals with ARCL1C, in which pulmonary abnormalities seem to develop earlier and are more pronounced than in those with ARCL1B. Individuals with ARCL1B, on the other hand, display cardiovascular aberrations, involving arterial tortuosity, aneurysms and stenosis, which are not seen in ARCL1C patients (Callewaert and Urban, 2016;Loeys et al., 2015). Fibulin-4 R/R mice closely model ARCL1B, including severe cardiovascular abnormalities (Hanada et al., 2007;Moltzer et al., 2011). Contrary to Ltbp4 −/− mice, which resemble the features of ARCL1C, Ltbp4S −/− mice develop a milder form of ARCL1C with a later onset of symptoms and prolonged survival (Bultmann-Mellin et al., 2015). The generated Ltbp4S −/− ; Fibulin-4 R/R mice displayed very pronounced symptoms of both ARCL1B and ARCL1C, indicating the interdependence between Ltbp-4L and fibulin-4.

SDS-PAGE and immunoblotting
Protein expression levels were determined by western blotting, using SDS-PAGE as previously described (Bultmann et al., 2013). All primary antibodies are listed in Table S2.

Histology and immunohistochemistry
Tissue architecture and localization of proteins were analyzed using previously described histological and immunohistochemical stainings (Bultmann-Mellin et al., 2015). All primary antibodies are listed in Table S2.

Statistical evaluation
Data are presented as mean±s.d. Differences between groups were analyzed by log-rank test or two-way ANOVA, followed by Bonferroni correction as appropriate. Statistical significance of post-hoc analyses were defined as P-values of *P<0.05 and **P<0.01. Calculations were performed using SPSS22 (IBM Deutschland, Ehningen, Germany).