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Title: Mechanisms underlying the differential regulation of vascular smooth muscle cell and endothelial cell profileration in response to cyclic AMP
Author: Hewer, Richard C.
ISNI:       0000 0004 2718 6095
Awarding Body: University of Bristol
Current Institution: University of Bristol
Date of Award: 2011
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Proliferation of vascular smooth muscle cells (VSMC) and endothelial cells (EC) is an important aspect of vascular pathologies. VSMC proliferation contributes to pathological intimal thickening and plays a role in the failure of clinical interventions to treat vascular disease. Surgical interventions can result in endothelial denudation, and so EC proliferation is necessary to re-establish effective endothelial barrier function. However, current treatments used to combat VSMC proliferation are non-selective, also affecting EC proliferation. There is a clear need to develop cell-selective anti-proliferative therapies for the treatment of vascular disease. To this end, this study set out to gain a deeper understanding of the mechanisms controlling proliferation of VS MC and EC. The anti-mitogenic properties of cyclic AMP (cAMP) in VSMC are well established. However, the effect of cAMP on EC proliferation is less clear, with conflicting reports in the literature. This study characterised the proliferative response of VSMC and EC to cAMP. As expected, cAMP elevation in VSMC resulted in G1 arrest. Interestingly, cAMP inhibited EC proliferation in a density-dependent manner; G1 arrest was observed in near-confluent cultures but not in sparse cultures. cAMP elevation in VSMC also induced arborisation (stellate morphology) including loss of actin stress fibres and focal adhesions, consistent with previous reports. cAMP- induced stellate morphology observed in VSMC does not occur in EC. Integrity of the actin cytoskeleton is a critical factor in cell cycle progression. Therefore, this study tested the hypothesis that disruption of the actin cytoskeleton plays a mechanistic role in VSMC growth arrest induced by cAMP. The role of Rho GTPases, well-known actin cytoskeleton regulators, was investigated. Interestingly, activity of RhoA was inhibited by cAMP in VSMC and EC, despite little effect on the actin cytoskeleton in EC. Overexpression of constitutively active RhoA significantly reversed cAMP-induced G1 arrest in VSMC and EC, indicating an essential role for RhoA in these effects. It is currently unclear why cAMP induces stellate morphology in VSMC but not in EC. Density-dependent inhibition of EC proliferation by cAMP suggested a mechanistic role for cell-cell contact. Involvement of the adherens junction protein VE-cadherin was tested. However, siRNA-mediated knockdown of VE-cadherin or lack-of- function mutations could not prevent cAMP-induced G1 arrest in EC, ruling out any involvement of VE-cadherin in this effect. Protein Kinase A (PKA) is well-recognised as a mediator of cAMP signalling. The last decade has seen considerable interest in a novel cAMP-sensitive protein, Epac (Exchange factor activated by cAMP). However, the role of Epac in regulating VSMC proliferation was unknown. This study demonstrated that Epac and PKA both play an essential role in cAMP-induced Gl arrest and stellate morphology of VSMC. Surprisingly though, the role of Epac in these effects was not mediated by its well- characterised effector, the ras-like GTPase Rapl. These data provide interesting new insights into the mechanisms controlling VSMC and EC proliferation and contribute to the future development of VS MC-selective anti-proliferative therapies.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available