Effects of The Acta2 R258C Mutation On Vascular Smooth Muscle Cell Phenotype and Properties

<p>Thoracic Aortic Aneurysms and Dissections (TAAD) are the fifteenth leading cause of death in the United States. About 15% of TAAD patients have family history of the disease. The most commonly mutated gene in these families is <em>ACTA2</em>, encoding smooth muscle-specific α-actin. <em>ACTA2</em> missense mutations predispose individuals both to TAAD and to vascular occlusive disease of small, muscular arteries.</p> <p>Mice carrying an <em>Acta2 </em>R258C mutant transgene with a wildtype <em>Acta2 </em>promoter were generated and bred with <em>Acta2^-/- </em>mice to decrease the wildtype: mutant <em>Acta2 </em>ratio. <em>Acta2^{+/+ R258C TG}</em>mice have decreased aortic contractility without aortic disease. <em>Acta2^{+/- R258C TG} </em>mice, however, have significant aortic dilatations by 12 weeks of age and a hyperproliferative response to injury. We characterized smooth muscle cells (SMCs) from bothmouse models under the hypothesis that mutant α-actin has a dominant negative effect, leading to impaired contractile filament formation/stability, improper focal adhesion maturation and increased proliferation.</p> <p>Explanted aortic SMCs from <em>Acta2^{+/+ R258C TG}</em> mice are differentiated - they form intact filaments, express higher levels of contractile markers compared to wildtype SMCs and have predominantly nuclear Myocardin-Related Transcription Factor A (MRTF-A) localization. However, ultracentrifugation assays showed large unpolymerized actin fractions, suggesting that the filaments are brittle. In contrast, <em>Acta2^{+/- R258C TG}</em> SMCs are less well-differentiated, with pools of unpolymerized actin, more cytoplasmic MRTF-A and decreased contractile protein expression compared to wildtype cells. Ultracentrifugation assays after treating <em>Acta2^{+/- R258C TG}</em>SMCs with phalloidin showed actin filament fractions, indicating that mutant α-actin can polymerize into filaments.</p> <p>Both <em>Acta2^{+/+ R258C TG}</em>and <em>Acta2^{+/- R258C TG}</em>SMCs have larger and more peripheral focal adhesions compared to wildtype SMCs. Rac1 was more activated in <em>Acta2^{+/+ R258C TG}</em>SMCs; both Rac1 and RhoA were less activated in <em>Acta2^{+/- R258C TG}</em> SMCs, and FAK was more activated in both transgenic SMC lines compared to wildtype. Proliferation in both cell lines was significantly increased compared to wildtype cells and could be partially attenuated by inhibition of FAK or PDGFRβ. These data support a dominant negative effect of the <em>Acta2 </em>R258C mutation on the SMC phenotype, with increasing phenotypic severity when wildtype: mutant α-actin levels are decreased.</p>

Name thesis:degree_name

Masters of Science (MS)

Level thesis:degree_level

Thesis (MS)

Year dc:date.available

2012

Author dc:creator

• Byanova, Katerina L

Contributors dc:contributor

• Dianna M. Milewicz, MD, PhD
• Fernando Cabral, PhD
• Guangwei Du, PhD

• TAAD
• smooth muscle
• ACTA2
• Medicine and Health Sciences

Repository record dc:identifier

https://digitalcommons.library.tmc.edu/utgsbs_dissertations/248

OAI identifier oai:identifier

oai:digitalcommons.library.tmc.edu:utgsbs_dissertations-1304