Cloning and characterisation of genes differentially expressed during smooth muscle phenotypic modulation

The precise factors that regulate and control myointimal thickening in\ua0arteries are incompletely understood. Many proposed mechanisms are based on\ua0a defect or injury to the endothelium resulting in change in phenotype of vascular\ua0smooth muscle cells followed by their migration, proliferation, synthesis of matrix\ua0and accumulation of lipid. The SMC involved in atherogenesis or after balloon\ua0catheter injury are thus phenotypically different from SM C of the normal,\ua0undiseased blood vessel.The aims of this thesis are: 1) to study the early response of gene(s) to\ua0vascular injury; 2) compare the expression of genes involved in response to\ua0injury of SMC both in vivo and in vitro; 3) to clone and characterise genes\ua0differentially expressed in myointimal thickening after balloon deendothelialisation;\ua0and 4) to further characterise proteins previously implicated in\ua0SMC phenotypic change, including those involved in heparan sulphate\ua0metabolism.A differential display technique was employed to identify and isolate the\ua0genes that are regulated during the development of an injury induced myointimal\ua0thickening. Balloon catheter injury was performed on the aorta of 14 week old\ua0rabbits which were sacrificed at 6 hours, 4, 7 and 28 days after injury. RNA was\ua0isolated from these aortae and from that of an uninjured control. RNA was also\ua0isolated from rabbit aortic primary SMC cultures at 2 and 4 days after seeding.\ua0cDNA was synthesised from both sources of RNA and compared on a 6%\ua0acrylamide gel. A total of 17 differentially expressed fragments were excised\ua0from the acrylamide gel.The fragments were re-amplified and 11 were successfully cloned.\ua0Ranging from 100 to 500 bp, the clones were sequenced and further\ua0investigated. Restriction map analysis was carried out for each clone along with\ua0a database search to identify any of the clones. High sequence homology to\ua0sequences in the database included matches with the long interspersed repeats\ua0L10c4, L10c5, human non muscle myosin heavy chain mRNA, phospholipase\ua0A2 receptor precursor, while the remaining clones were novel with no high\ua0identity matches with sequences in the GenBank database.RT-PCR was used to monitor the differential expression of each clone\ua0both in vivo and in vitro. The expression of each transcript was quantified\ua0against GAPDH. The expression of most clones decreased immediately 6 hours\ua0after balloon de-endothelialisation, while some regained or exceeded expression\ua0levels 28 days after balloon injury compared to those levels found in the normal\ua0artery. RT-PCR was also used to monitor expression levels of three previously\ua0identified genes - tropomyosin, rpL17 and NST.With the knowledge of the expression levels, in situ hybridisation was\ua0used to observe the localisation of each transcript in the normal and injured\ua0artery. The further characterisation of tropomyosin and NST by Western blotting\ua0was reported. Protein levels were quantified in the normal artery and artery\ua0injured 6,24 hours, 3, 5, 7,14 and 28 days previously.As the degradation of HSPG plays a major role in the phenotypic\ua0modulation of SMCs, a simple method of its detection was developed involving\ua0SDS PAGE and Alcian blue. This methodology was then used to measure\ua0increased heparanase production induced by IFN‐γ treated macrophages.This study has found 11 genes, the majority of which are novel, that are\ua0differentially expressed during the process of myointimal thickening and SMC\ua0phenotypic modulation. The characterisation of these genes may lead to\ua0strategies to prevent excess thickening which can occur following balloon\ua0angioplasty to remove primary atherosclerotic plaques