The stellate vascular smooth muscle cell phenotype is induced by IL-1β via the secretion of PGE2 and subsequent cAMP-dependent protein kinase A activation

Abstract

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.

Intracellular cAMP Sensor EPAC: Physiology, Pathophysiology, and Therapeutics Development

“…The high-resolution imaging of cAMP dynamics in single living cells offers researchers the opportunity to not only determine the absolute free cAMP concentrations in living cells with high temporal and spatial resolution (95,900), but to also address several important unanswered questions involving cAMP signaling, in particular potential cross-talk mechanisms with other signaling pathways, as well as the roles of various GPCRs (3,85,1067,1129), G proteins (553,672), ACs (1113), and PDEs (789,1023) in controlling intracellular cAMP dynamics in response to distinct stimuli (383,408,698,1077,1185). For example, cAMP and Ca 2ϩ signaling pathways are known to be interconnected spatially and temporally in eukaryote cells (202,357).…”

Ending Restenosis: Inhibition of Vascular Smooth Muscle Cell Proliferation by cAMP

“…Cyclic-AMP induced growth arrest in VSMC is associated with a profound but reversible change in cell morphology resulting from reorganisation of the actin cytoskeleton [32,79,94,177,178,179,180,181]. Stimulation of VSMC with forskolin, cAMP analogues or activation of the prostacyclin, adenosine A2B receptors or β-adrenergic receptors, results in a rapid acquisition of a ‘stellate’ morphology, characterised by a condensation of the cell body and formation of long branching processes.…”

Novel short isoforms of adenylyl cyclase as negative regulators of cAMP production

“…After annealing oligo(dT) primers (1 µM) to template RNAs (0.5-1 µM) at 70°C for 5 min, we initiated primer extension by adding M-MLV reverse transcriptase plus 0.5 mM dNTPs, 1 unit of RNaseOUT recombinant ribonuclease inhibitor and 20 mM DTT, and incubated the reaction mixture for a further 60 min at 37°C. Quantitative PCR was performed with a LightCycler 480 instrument as described previously [24]. The forward and reverse primers used for selective amplification of the cDNAs 8 encoding rat cyclophilin A and ACs are presented in Table S1.…”

“…Cells on collagen (VSMCs) or poly-L-lysine (HEK-293)-coated coverslips were infected with the T Epac VV -encoding adenoviral vector (~100 particles per VSMC) or transfected with the T Epac VV -encoding vector (HEK-293) for 24 h. The generation of the T Epac VV -encoding adenovirus has been described elsewhere [24]. Coverslips were placed in a microscope chamber continually perfused (2 mL.min -1 ) with a buffer containing 125 mM NaCl, 2 mM CaCl 2 , 1 mM MgCl 2 , 1.25 mM NaH 2 PO 4 , 26 mM NaHCO 3 , 25 mM glucose, maintained at 32°C and saturated with 95% O 2 -5% CO 2 .…”