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

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.

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.

Treatment with vitamin k(2) combined with bisphosphonates synergistically inhibits calcification in cultured smooth muscle cells.

AIM: Vascular calcification is a common feature in patients with advanced atherosclerosis, postmenopausal women and patients with renal failure, which results in reduced elasticity of arteries. Pamidronate, a bisphosphonate, is used as a therapeutic agent for anti-osteoporosity, although there are adverse side effects, such as renal damage and aortic inflamed plaque rupture. In the present study, we demonstrated the effects of vitamin K(2) alone or in combination with pamidronate in an arterial calcification model induced using inorganic phosphate in cultured bovine aortic smooth muscle cells (BASMCs). METHODS: Calcification was induced by the addition of Pi (3 mM) in BASMCs. Calcium deposition was determined by Calcium C-test Wako and von Kossa staining. mRNA expression was assessed by semi-quantitative reverse transcription-polymerase chain reaction. RESULTS: Calcium deposition assay and von Kossa staining showed that calcification could be inhibited in a dose-dependent manner by treatment with vitamin K(2) alone, and that its inhibitory effect was enhanced when combined with pamidronate. It was found that the expression of tropoelastin mRNA was synergistically enhanced by combined treatment with vitamin K(2) and pamidronate, and the expression matrix Gla protein mRNA and osteopontin mRNA expression were also enhanced and suppressed, respectively, by treatment with vitamin K(2) or pamidronate. Moreover, our data showed that the suppression of TE expression by siRNA significantly increased Pi-induced vascular calcification. CONCLUSION: Taken together, our study suggests that vitamin K(2) in combination with pamidronate synergistically inhibits arterial calcification via the increased expression of tropoelastin, which would be a useful marker for developing effective therapeutic or prophylactic agents for arterial calcification.

Accelerated calcification represses the expression of elastic fiber components and lysyl oxidase in cultured bovine aortic smooth muscle cells.

Vascular calcification is a common feature of advanced atherosclerosis resulting in reduced elasticity of elastic arteries. However, the relationship between elastic fibers and vascular calcification at the molecular and cellular levels remains unknown. We investigated the expression of major elastic fiber components such as tropoelastin (TE) and fibrillin-1 (FBN1) and elastin-related enzyme, lysyl oxidase (LO), in a calcification model using beta-glycerophosphate (beta-GP) in cultured bovine aortic smooth muscle cells (BASMCs). Ten mM of beta-GP stimulated calcium deposition in a time-dependent manner. As determined by Western blot analysis, 10 mM of beta-GP time-dependently decreased TE and FBN1 protein levels. TE, FBN1, and LO mRNA levels, assessed by reverse transcription-polymerase chain reaction, were also decreased by exposure to 10 mM beta-GP. Furthermore, we investigated whether the processes of calcification in BASMCs directly control these regulations. In experiments using levamisole, an alkaline phosphatase inhibitor, and DMDP, a bisphosphonate, both inhibitors inhibited down-regulation during beta-GP-induced calcification, suggesting that the down-regulation of TE, FBN1, and LO directly relates to calcium deposition. In cases of vascular calcification, the decreased expression of TE, FBN1, and LO may be partially responsible for decreased vascular elasticity and also for the decreased formation of new elastic fibers.

Characterization of an in vitro model of calcification in retinal pigmented epithelial cells.

Little is known about the relationship at the molecular and cellular levels between vascular calcification and elastic fibers essential for elasticity. To gain a better understanding of the physiological function of elastin in vascular calcification, we developed a calcification model on cultured bovine retinal-pigmented-epithelial cells (RPEs) that do not express endogenous tropoelastin. The addition of inorganic phosphate (NaH2PO4; Pi) induced calcium deposition in RPEs. The Pi-induced calcification, as assessed by the o-cresolphthalein complexone method, Goldenbergs method, and von Kossa staining, was completely inhibited by treatment with clodronate (DMDP) and phosphonoformic acid (PFA) and was weakly suppressed by treatment with levamisole. Moreover, the osteopontin mRNA expression was upregulated in the Pi-induced calcification of RPEs. These reactions in RPEs were characteristically consistent with those already established in cultured bovine aortic smooth muscle cells (BASMCs). Furthermore, bacterially expressed tropoelastin inhibited calcium deposition in RPEs as well as in BASMCs. Finally, Pi-induced calcification was partially suppressed after the addition of tropoelastin due to elastic fiber formation. In conclusion, we suggest that this calcification model in RPEs is useful for analyzing the relation between elastic fibers and vascular calcification, and that tropoelastin and elastic fibers may contribute to the inhibition of vascular calcification.

Tropoelastin inhibits vascular calcification via 67-kDa elastin binding protein in cultured bovine aortic smooth muscle cells.

In cases of vascular calcification, the expression of tropoelastin is down-regulated, which most likely decreases elastic fiber formation. However, the function of tropoelastin in vascular calcification remains unknown. We investigated whether tropoelastin affects the induction of vascular calcification. Calcification was induced using inorganic phosphate in cultured bovine aortic smooth muscle cells. The increase in tropoelastin due to the addition of recombinant bovine tropoelastin (ReBTE; 1 or 10 microg/ml) or beta-aminopropionitrile (25 microg/ml) significantly inhibited calcification at day 6, as assessed by the o-cresolphthalein complexone method. The addition of an elastin-derived peptide, VGVAPG peptide (0.1-1,000 nM), inhibited calcification at day 6 in a dose-dependent manner. In addition, these responses of beta-aminopropionitrile, ReBTE, and VGVAPG peptide were confirmed using von Kossa staining. To examine whether ReBTE inhibited calcium deposition via the elastin binding protein, lactose and elastin-specific antibody were used. The combination of lactose (20 mM) or this antibody (50 microg/ml) with ReBTE (10 microg/ml) attenuated the inhibition of calcification. These results suggest that increased tropoelastin inhibits vascular calcification in this model via the interaction between tropoelastin and elastin binding protein.

Upregulation of rhoA mRNA in bronchial smooth muscle of antigen-induced airway hyperresponsive rats.

It has recently been suggested that RhoA plays an important role in the enhancement of Ca2+ sensitization observed in smooth muscle contraction. In the present study, the expression of rhoA mRNA in the bronchial smooth muscle of antigen-induced airway hyperresponsive rats was compared with that of control animals. Reverse transcription-polymerase chain reaction experiments using total RNA from these tissue specimens and the specific primers revealed rhoA mRNA to be expressed in bronchial smooth muscle of the rat. The rhoA mRNA expression in bronchial smooth muscle of the hyperresponsive rats was significantly increased in comparison to that of control animals. It is thus possible that upregulation of RhoA protein might be involved in the mechanism underlying the increased contractility of the bronchial smooth muscle which occurs with airway hyperresponsiveness.