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Development 130 (2): 411-423

A critical role for elastin signaling in vascular morphogenesis and disease

Satyajit K. Karnik1,2,*, Benjamin S. Brooke1,2,*, Antonio Bayes-Genis3,*, Lise Sorensen1, Joshua D. Wythe1,2, Robert S. Schwartz4, Mark T. Keating5, and Dean Y. Li1,2,{dagger}

1 Program in Human Molecular Biology and Genetics, University of Utah, Salt Lake City, UT, USA
2 Department of Medicine and Oncological Science, University of Utah, Salt Lake City, UT, USA
3 Hospital Sant Pau, Barcelona, Spain
4 Minnesota Cardiovascular Research Institute, Minneapolis, MN, USA
5 Howard Hughes Medical Institute, Department of Cell Biology, Harvard Medical School, Department of Cardiology, Children's Hospital, Boston, MA, USA



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Fig. 1. Elastin inhibits proliferation of vascular smooth muscle cells. (A) RT-PCR experiments confirm that murine vascular smooth muscle cells lines express cell specific markers. The elastin gene product is detected in Eln+/+ and not Eln-/- cells. Immunofluorescence analysis with elastin antibodies and accompanying phase photomicrographs demonstrate that Eln+/+ vascular smooth muscle cells (B) synthesize and secrete elastin matrix, whereas Eln-/- vascular smooth muscle cells (C) do not produce elastin. (D) Assay measuring cell numbers demonstrates that Eln-/- vascular smooth muscle cells proliferate at a much higher rate than do Eln+/+ vascular smooth muscle cells (P<0.0001, ANOVA). This difference is eliminated by the addition of recombinant elastin gene product or tropoelastin (tEln). (E) Assay measuring [3H]thymidine incorporation assay demonstrates that Eln-/- vascular smooth muscle cells proliferate at a rate over twofold greater than that for Eln+/+ cells. This difference is eliminated by the inhibition of Eln-/- cells proliferation by tropoelastin (P<0.0001, ANOVA).

 


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Fig. 2. Elastin induces a mature contractile phenotype in vascular smooth muscle cells. (A-E) Immunofluorescence analysis for SM {alpha}-actin reveals that Eln+/+ vascular smooth muscle cells (A) have a highly organized network of actin stress fibers, a hallmark of mature contractile vascular smooth muscle cells. By contrast, there is a paucity of actin stress fibers in Eln-/- vascular smooth muscle cells (B; outlined by white dots). The elastin gene product, recombinant tropoelastin, induces the formation of organized actin stress fibers in Eln-/- vascular smooth muscle cells (D), but does not affect Eln+/+ vascular smooth muscle cells (C). Scoring analysis demonstrates a significant increase in the percentage of Eln-/- vascular smooth muscle cells with organized actin myofilaments after elastin treatment (P<0.0001) (E). Tropoelastin mediated actin polymerization is unaffected when Eln-/- cells are treated with either a gene transcription inhibitor, actinomycin D, or a protein translation inhibitor, cycloheximide. The Rho kinase inhibitor, Y27632, blocks actin polymerization of tropoelastin treated Eln-/- cells. (F-J) Immunofluorescence analysis for vinculin reveals that Eln+/+ vascular smooth muscle cells (F) have well-defined focal adhesions (arrowheads). By contrast, Eln-/- vascular smooth muscle cells (G; outlined by white dots) have poorly defined focal adhesions. Tropoelastin induces well-defined focal adhesions (arrows) throughout Eln-/- vascular smooth muscle cells (I), but does not affect Eln+/+ vascular smooth muscle cells (H). Scoring analysis revealed a significant increase (P<0.0001) in the percentage of Eln-/- vascular smooth muscle cells with defined focal adhesions after tropoelastin treatment (J). (K,L) Immunofluorescence staining with antisera against tubulin reveals no difference between Eln+/+ vascular smooth muscle cells (K) and Eln-/- vascular smooth muscle cells (L). Exposure times for all images are the same. Results represent the mean±s.d. from three individual experiments.

 


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Fig. 3. Elastin controls migration of vascular smooth muscle cells. (A) Eln+/+ and Eln-/- vascular smooth muscle cells migrate to tropoelastin in a concentration-dependent manner, indicating that tropoelastin is a specific and direct stimulus for vascular smooth muscle cell localization. In addition, Eln-/- cells consistently migrated at a higher rate than Eln+/+ cells, suggesting that the autocrine production of elastin by Eln+/+ cells reduces their chemotaxis to external stimuli. (B) Tropoelastin inhibits vascular smooth muscle cell migration to the chemotactic growth factor PDGF. A modified Boyden chamber assay was used for all experiments to determine the total number of migrated cells in 15 randomly selected high power microscope fields (HPF). Data shown are the mean±s.e.m. from three independent wells.

 


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Fig. 4. Tropoelastin regulates migration and actin polymerization via a non-integrin, G-protein-coupled signaling pathway. (A) Eln-/- vascular smooth muscle cells migrate to recombinant tropoelastin in an EDTA insensitive manner. By comparison, we show that vascular smooth muscle cell migration to collagen, which is mediated by integrins, is EDTA sensitive. (B) Tropoelastin mediated actin polymerization of Eln-/- cells is EDTA insensitive. (C) Migration of Eln-/- vascular smooth muscle cells towards tropoelastin is pertussis toxin sensitive. Control experiments with the B protomer of pertussis toxin alone demonstrate that the effect of pertussis toxin is specific to its ability to inhibit G-protein signaling. The migration of Eln-/- cells to PDGF (30 ng/ml) is insensitive to pertussis toxin. This control experiment demonstrates that Eln-/- cells treated with pertussis toxin can respond normally to stimuli other than tropoelastin. (D) Tropoelastin-mediated actin polymerization of Eln-/- vascular smooth muscle cells is pertussis toxin sensitive. Control experiments demonstrate that the B protomer of pertussis toxin does not inhibit actin polymerization. Together, these experiments indicate that tropoelastin regulates vascular smooth muscle cells through a pertussis toxin-sensitive G-protein signaling pathway. (E) Cholera toxin (ctxn) and forskolin (frskln) increase the baseline levels of cAMP in Eln-/- vascular smooth muscle cells by constitutive activation of the Gs pathway. In the presence of tropoelastin and forskolin, there is a marked decrease in cAMP. A similar decrease in cAMP levels is observed when tropoelastin is added to cholera toxin pretreated cells. The reduction in cAMP was blocked by pertussis toxin and not B-protomer indicating a Gi specific pathway. These experiments indicate that tropoelastin activates a receptor that signals through Gi to inhibit adenylate cyclase, and reduce cAMP levels. (F) Tropoelastin activates RhoA in a pertussis toxin-sensitive manner. Co-immunoprecipitation experiments demonstrate that tropoelastin treatment of Eln-/- cells results in a marked elevation of activated RhoA. This response is pertussis toxin sensitive and B-protomer insensitive indicating that tropoelastin activation of the Rho pathway requires Gi activity. (G) Proposed molecular mechanism for tropoelastin-mediated actin polymerization in vascular smooth muscle cells.

 


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Fig. 5. Elastin therapy reduces vascular smooth muscle cell accumulation and arterial obstruction in vivo. (A) Scanning electron micrographs of elastin sheaths. Scale bar: 10 µm. (B) Elastin matrix sheath covering an expanded metal stent. (C,D) Representative cross-sections taken from control arteries (n=14) display the development of a thick fibrocellular neointima (C), whereas the neointima is substantially decreased in elastin sheath (arrows) treated arteries (n=13) with the same injury score (D). (E,F) Over a range of injury, the percent lumen stenosis (E) and mean neointimal thickness (F) were significantly reduced (both P<0.0001) in injured porcine coronary arteries treated with elastin sheath-stents compared with control stents.

 


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Fig. 6. Model of elastin-vascular smooth muscle cell interactions in development and disease. (A) During normal development, vascular smooth muscle cells synthesize and secrete elastin polymers that form concentric rings of elastic lamellae around the arterial lumen. Elastin provides mechanical support to the vessel wall, and signals vascular smooth muscle cells to localize around the elastic lamellae and remain in a quiescent, contractile state. (B) In the absence of elastin, this morphogenic signal is lost resulting in pervasive subendothelial migration and proliferation of vascular smooth muscle cells that occludes the vascular lumen. (C) This leads us to propose that the focal disruption and/or destruction of elastin in the mature artery by vascular injury releases smooth muscle cells to dedifferentiate, migrate and proliferate, and contributes to neointimal formation.

 


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