Atherosclerosis development is associated with morphological changes to intimal cells, leading to a stellate cell phenotype. In this study, we aimed to determine whether and how key pro-atherogenic cytokines present in atherosclerotic plaques (IL-1β, TNFα and IFNγ) could induce this phenotype, as these molecules are known to trigger the transdifferentiation of vascular smooth muscle cells (VSMCs). We found that, IL-1β was the only major inflammatory mediator tested capable of inducing a stellate morphology in VSMCs. This finding was confirmed by staining for F-actin and vinculin at focal adhesions, as these two markers were disrupted only by IL-1β. We then investigated the possible association of this IL-1β-dependent change in morphology with an increase in intracellular cAMP concentration ([cAMP]), using the FRET-based biosensor for cAMP (T)Epac(VV). Experiments in the presence of IL-1β or medium conditioned by IL-1β-treated VSMCs and pharmacological tools demonstrated that the long-term increase in intracellular cAMP concentration was induced by the secretion of an autocrine/paracrine mediator, prostaglandin E₂(PGE₂), acting through the EP4 receptor. Finally, by knocking down the expression of the regulatory subunit PKAR1α, thereby reproducing the effects of IL-1β and PGE₂ on VSMCs, we demonstrated the contribution of PKA activity to the observed behavior of VSMCs.
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