Injury-induced activation of vascular smooth muscle cells. Role of specific gene expression and intracellular signalling pathways

Abstract: Migration and proliferation of smooth muscle cells (SMC) are important events in the development of atherosclerosis and restenosis following angioplastic surgery. It is important to gain further knowledge about the mechanisms regulating SMC form and function in order to understand the underlying disease process. In this thesis I have used cultured rat aortic SMC and analysed how these cells respond to either a mechanical or enzymatic injury. The objective has been to identify the molecular mechanisms involved in injury-induced activation of SMC. I have focused my studies on the role of molecules regulating migration and proliferation. These include metalloproteinases and the intracellular signalling molecule ERK1/2. Increased expression of the migration-related stromelysin gene as well as early and transient expression of the transcription factor Ets-1 were detected when primary SMC were established in culture. Up-regulated Ets-1 expression was also seen in the rat carotid artery following balloon-injury. Since Ets-1 regulates the stromelysin gene, the role of stromelysin in injury-induced SMC activation was further investigated using antisense oligonucleotides directed towards the stromelysin translation initiation site. Treatment with antisense oligos inhibited phenotypic change as well as decreased the migration and proliferation of SMC after injury in vitro. Furthermore, these antisense oligos reduced neointimal thickening after balloon-injury in rat carotid arteries. Injury-induced ERK1/2 phosphorylation could be coupled to altered SMC function by the use of the MEK1-inhibitor PD98059. In addition, the synthetic metalloproteinase inhibitor Batimastat (BB94) reduced ERK1/2 activation as well as SMC migration and proliferation. Further studies demonstrated that Ca2+, calmodulin, tyrosine kinases, G-proteins and Src all appear to be involved in injury-induced ERK1/2 activation, migration and proliferation in SMC. In conclusion, these results suggest (1) a role for Ets-1 and metalloproteinases such as stromelysin in the SMC response to injury, (2) that the ERK1/2 is a key signalling molecule in the alteration of SMC function, (3) that release of Ca2+ is necessary for optimal ERK1/2 activation, and (4) that SMC activation due to injury is a result of signal transmission via several converging ERK1/2 connected pathways.