O22	The effects of cyclic strain on the cytoskeleton of vascular smooth muscle cells.
1A.Halka, 2N.Turner, 1M.Murphy, 1J.Ghosh, 1N.Khwaja, 1A.Carter, 2C.Kielty, 1M.Walker
1Department of Vascular Surgery, Manchester Royal Infirmary, Manchester, GB; 2UK Centre for Tissue Engineering, University of Manchester, Manchester, GB.

Introduction In embryos vascular smooth muscle cells (VSMC) adopt a synthetic phenotype and contribute to angiogenesis. With maturation VSMC assume a contractile phenotype enhancing vascular tone and contractility. Excessive stretch of blood vessels occurs during hypertension and in grafted autologous conduits. This increases the incidence of myointimal hyperplasia (MIH) that causes bypass graft failure. MIH occurs when VSMC pathologically alter their phenotype from contractile to synthetic. Methods Human coronary artery smooth muscle cells from a 19 year old male were obtained commercially. They were cultured on silicone membranes coated with Collagen IV, Laminin or ProNectin. Once above 95% confluent they were incubated in serum free medium to achieve quiescence. They were treated with biaxial cyclic strain using the Flexercell System for two or 48 hours. Two types of strain: physiological and pathological and two different wave-forms: sine and cardiac were used. Experiments were also performed in culture medium containing serum. After manipulation VSMC were prepared for immunofluorescence or had their mRNA extracted. This was examined by employing the reverse transcriptase polymerase chain reaction (RT-PCR) using primers for the VSMC contractile markers: SM α actin, calponin and SM myosin heavy chain. Results The coating on which the VSMC were cultured influenced their appearance. The VSMC orientation was affected by the direction of strain. When exposed to physiological strain the VSMC cytoskeleton was better organised than was the case with cells treated with pathological stretch. With physiological stretch RT-PCR demonstrated enhanced expression of contractile markers whilst the converse occurred in VSMC exposed to pathological stretch. The VSMC structure was also affected by different rhythms. Conclusions It is possible to manipulate VSMC phenotype. This would enable tissue engineers to ensure the presence of VSMC that would be appropriate to the task that they are required to perform, be it synthetic or contractile. By seeding these cells onto engineered grafts the development of MIH may be avoided.

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