Role of extracellular matrix in vascular smooth muscle cell behaviour and the identification of novel markers of cell phenotype.

Carragher, Neil (1996). Role of extracellular matrix in vascular smooth muscle cell behaviour and the identification of novel markers of cell phenotype. PhD thesis The Open University.



Increased synthesis and deposition of extracellular matrix proteins in the blood vessel wall are implicated in vascular disorders such as atherosclerosis, restenosis and hypertension. (Liau and Chan 1989). The increase in extracellular matrix proteins can be mostly attributed to smooth muscle cells (SMC’s) within vascular lesions (Mecham et al 1987). It is presumed that modulation of SMC’s from their normally quiescent, contractile phenotype to a proliferative synthetic phenotype results in increased synthesis of extracellular matrix proteins. Studies have also demonstrated that signals elicited from the extracellular matrix (ECM) may play a role in the regulation of this SMC phenotypic modulation (Yamamoto et al 1993; Hedin et al 1988). The mechanism by which ECM can alter the phenotypic state of SMC’s is not well understood but clearly involves the induction of intracellular signals as a consequence of ECM ligand - cell surface integrin binding. These signals must subsequently exert downstream molecular events altering gene expression and ultimately cell phenotype.

The research project presented in this thesis examined the influence various extracellular matrix substrates have on vascular SMC behaviour in vitro. Initial observations demonstrated that SMC’s cultured on different matrix substrates exhibit distinct morphological growth patterns. Functional differences in SMC proliferation and migration rates were also observed in response to seeding on different ECM surfaces.

Analysis of the expression levels of known SMC phenotypic protein markers between SMC’s cultured on different matrix substrates did not reveal any significant differences in protein expression. Slight upregulation of Myosin Light Chain Kinase (MLCK)-210 kd isoform was observed in SMC’s cultured on cellular fibronectin, Collagen III and Vitronectin substrates. The Meta-vinculin protein was upregulated in SMC’s cultured on fibronectin coated substrates.

In order to identify altered gene expression patterns induced by ECM adhesion, SMC populations cultured on fibronectin coated plastic and SMC’s grown on uncoated plastic were selected for analysis by the differential display technique. Differential display is a recently developed PCR based technique that allows the identification of differentially expressed genes between related cell populations (Liang and Pardee 1992). Much effort was spent optimising the differential display procedure to overcome such limitations as primer redundancy and high false positive selection rates. However, as a result of the persistence of false positives only one gene, meta-vinculin was confirmed as being differentially expressed between SMC populations cultured on fibronectin coated and uncoated tissue culture plastic surfaces.

It was concluded that the failure to identify a significant number of fibronectin modulated genes was probably a result of limitations in the differential display procedure as carried out in this study, and may also possibly be due to the existence of only very minor differences in phenotype between SMC’s grown on plastic with or without fibronectin precoating.

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