Christopher H. Ciliberto

Functional Rescue of Elastin Insufficiency in Mice by the Human Elastin Gene: Implications for Mouse Models of Human Disease

Diseases linked to the elastin gene arise from loss-of-function mutations leading to protein insufficiency (supravalvular aortic stenosis) or from missense mutations that alter the properties of the elastin protein (dominant cutis laxa). Modeling these diseases in mice is problematic because of structural differences between the human and mouse genes. To address this problem, we developed a humanized elastin mouse with elastin production being controlled by the human elastin gene in a bacterial artificial chromosome. The temporal and spatial expression pattern of the human transgene mirrors the endogenous murine gene, and the human gene accurately recapitulates the alternative-splicing pattern found in humans. Human elastin protein interacts with mouse elastin to form functional elastic fibers and when expressed in the elastin haploinsufficient background reverses the hypertension and cardiovascular changes associated with that phenotype. Elastin from the human transgene also rescues the perinatal lethality associated with the null phenotype. The results of this study confirm that reestablishing normal elastin levels is a logical objective for treating diseases of elastin insufficiency such as supravalvular aortic stenosis. This study also illustrates how differences in gene structure and alternative splicing present unique problems for modeling human diseases in mice.

The importance of elastin to aortic development in mice.

Elastin is an essential component of vertebrate arteries that provides elasticity and stores energy during the cardiac cycle. Elastin production in the arterial wall begins midgestation but increases rapidly during the last third of human and mouse development, just as blood pressure and cardiac output increase sharply. The aim of this study is to characterize the structure, hemodynamics, and mechanics of developing arteries with reduced elastin levels and determine the critical time period where elastin is required in the vertebrate cardiovascular system. Mice that lack elastin (Eln(-/-)) or have approximately one-half the normal level (Eln(+/-)) show relatively normal cardiovascular development up to embryonic day (E) 18 as assessed by arterial morphology, left ventricular blood pressure, and cardiac function. Previous work showed that just a few days later, at birth, Eln(-/-) mice die with high blood pressure and tortuous, stenotic arteries. During this period from E18 to birth, Eln(+/-) mice add extra layers of smooth muscle cells to the vessel wall and have a mean blood pressure 25% higher than wild-type animals. These findings demonstrate that elastin is only necessary for normal cardiovascular structure and function in mice starting in the last few days of fetal development. The large increases in blood pressure during this period may push hemodynamic forces over a critical threshold where elastin becomes required for cardiovascular function. Understanding the interplay between elastin amounts and hemodynamic forces in developing vessels will help design treatments for human elastinopathies and optimize protocols for tissue engineering.

Genetic modifiers of cardiovascular phenotype caused by elastin haploinsufficiency act by extrinsic noncomplementation.

Background: Variability in vascular pathology is associated with elastin loss-of-function mutations. Results: Quantitative trait loci and several candidate genes that modify vessel pathology were identified in a mouse model of elastin insufficiency. Conclusion: The effects of elastin insufficiency are determined by interactions between the primary elastin defect and unrelated secondary modifiers. Significance: Identification of modifier genes enhances our understanding of disease mechanisms associated with elastin mutations. Elastin haploinsufficiency causes the cardiovascular complications associated with Williams-Beuren syndrome and isolated supravalvular aortic stenosis. Significant variability exists in the vascular pathology in these individuals. Using the Eln+/− mouse, we sought to identify the source of this variability. Following outcrossing of C57Bl/6J Eln+/−, two backgrounds were identified whose cardiovascular parameters deviated significantly from the parental strain. F1 progeny of the C57Bl/6J; Eln+/−x129X1/SvJ were more hypertensive and their arteries less compliant. In contrast, Eln+/− animals crossed to DBA/2J were protected from the pathologic changes associated with elastin insufficiency. Among the crosses, aortic elastin and collagen content did not correlate with quantitative vasculopathy traits. Quantitative trait locus analysis performed on F2 C57; Eln+/−x129 intercrosses identified highly significant peaks on chromosome 1 (LOD 9.7) for systolic blood pressure and on chromosome 9 (LOD 8.7) for aortic diameter. Additional peaks were identified that affect only Eln+/−, including a region upstream of Eln on chromosome 5 (LOD 4.5). Bioinformatic analysis of the quantitative trait locus peaks revealed several interesting candidates, including Ren1, Ncf1, and Nos1; genes whose functions are unrelated to elastic fiber assembly, but whose effects may synergize with elastin insufficiency to predispose to hypertension and stiffer blood vessels. Real time RT-PCR studies show background-specific increased expression of Ncf1 (a subunit of the NOX2 NAPDH oxidase) that parallel the presence of increased oxidative stress in Eln+/− aortas. This finding raises the possibility that polymorphisms in genes affecting the generation of reactive oxygen species alter cardiovascular function in individuals with elastin haploinsufficiency through extrinsic noncomplementation.

Alternative splicing and tissue-specific elastin misassembly act as biological modifiers of human elastin gene frameshift mutations associated with dominant cutis laxa.

Background: A humanized mouse was developed to study elastin assembly and the pathogenesis of cutis laxa. Results: Mutant transcripts incorporate into elastic fibers of skin and lung with adverse effects but not aorta. Conclusion: Elastin frameshift mutations alter elastin assembly domains. Significance: The mechanism of elastic fiber assembly may not be the same in all tissues. Elastin is the extracellular matrix protein in vertebrates that provides elastic recoil to blood vessels, the lung, and skin. Because the elastin gene has undergone significant changes in the primate lineage, modeling elastin diseases in non-human animals can be problematic. To investigate the pathophysiology underlying a class of elastin gene mutations leading to autosomal dominant cutis laxa, we engineered a cutis laxa mutation (single base deletion) into the human elastin gene contained in a bacterial artificial chromosome. When expressed as a transgene in mice, mutant elastin was incorporated into elastic fibers in the skin and lung with adverse effects on tissue function. In contrast, only low levels of mutant protein incorporated into aortic elastin, which explains why the vasculature is relatively unaffected in this disease. RNA stability studies found that alternative exon splicing acts as a modifier of disease severity by influencing the spectrum of mutant transcripts that survive nonsense-mediated decay. Our results confirm the critical role of the C-terminal region of tropoelastin in elastic fiber assembly and suggest tissue-specific differences in the elastin assembly pathway.

Modification and functional inactivation of the tropoelastin carboxy-terminal domain in cross-linked elastin

The carboxy-terminus of tropoelastin is a highly conserved, atypical region of the molecule with sequences that define both cell and matrix interactions. This domain also plays a critical but unknown role in the assembly and crosslinking of tropoelastin during elastic fiber maturation. Using a competitive ELISA with an antibody to an elastase-resistant epitope in the carboxy-terminus of tropoelastin (domain-36), we quantified levels of the domain-36 sequence in elastase-derived peptides from mature, insoluble elastin. We found that the amount of carboxy-terminal epitope in elastin is approximately 0.2% of the expected value, assuming each tropoelastin monomer that is incorporated into the insoluble polymer has an intact carboxy-terminus. The low levels suggest that the majority of domain-36 sequence is either removed at some stage of elastin assembly or that the antigenic epitope is altered by posttranslational modification. Biochemical evidence is presented for a potential lysine-derived cross-link in this region, which would alter the extractability and antigenicity of the carboxy-terminal epitope. These results show that there is little or no unmodified domain-36 in mature elastin, indicating that the cell and matrix binding activities associated with this region of tropoelastin are lost or modified as elastin matures. A crosslinking function for domain-36 may serve to help register the multiple crosslinking sites in elastin and explains why mutations that alter the domain-36 sequence have detrimental effects on elastic fiber assembly.

Evidence for Heterozygous Abnormalities of the Elastin Gene (ELN) Affecting the Quantity of Vocal Fold Elastic Fibers: A Pilot Study

OBJECTIVES To investigate the effects of a heterozygous elastin gene (Eln) abnormality (deletion of one Eln allele) on the structural characteristics of the vocal fold lamina propria using a mouse model of human disease. STUDY DESIGN Cross-sectional between-subjects design. METHODS Five mice, four with heterozygous Eln deletions (Eln +/-) serving as an animal model for the human disease supravalvular aortic stenosis and one normal wild-type control (Eln +/+) were used for this study. Vocal folds were obtained from each animal and stained for the protein elastin using histochemical methods. Descriptive data from qualitative visual inspection and quantitative data from microscopic digital image analysis were collected to determine the staining density of elastic fibers within the vocal fold lamina propria. RESULTS Qualitative visual inspection revealed greater staining density (eg, a greater quantity) for elastic fibers in the Eln +/+ animal. Quantitative measurements using digital pixel analysis of staining density revealed significant differences between mice with the two genotypes, confirming the qualitative findings. CONCLUSIONS Results suggest that Eln requires two functioning alleles for normal structural development of the vocal fold lamina propria. This pilot evidence supports the hypothesis of a structural etiology causing altered vocal function in humans with a similar genotype.