Aspirin-triggered resolvin D1 attenuates PDGF-induced vascular smooth muscle cell migration via the cyclic adenosine monophosphate/protein kinase A (cAMP/PKA) pathway

Abstract

Background and objectivesResolvin D1 (RvD1) is a specialized pro-resolving lipid mediator that has been previously shown to attenuate vascular smooth muscle cell (VSMC) migration, a key process in the development of intimal hyperplasia. We sought to investigate the role of the cAMP/PKA pathway in mediating the effects of the aspirin-triggered epimer 17R-RvD1 (AT-RvD1) on VSMC migration.MethodsVSMCs were harvested from human saphenous veins. VSMCs were analyzed for intracellular cAMP levels and PKA activity after exposure to AT-RvD1. Platelet-derived growth factor (PDGF)-induced migration and cytoskeletal changes in VSMCs were observed through scratch, Transwell, and cell shape assays in the presence or absence of a PKA inhibitor (Rp-8-Br-cAMP). Further investigation of the pathways involved in AT-RvD1 signaling was performed by measuring Rac1 activity, vasodilator stimulated phosphoprotein (VASP) phosphorylation and paxillin translocation. Finally, we examined the role of RvD1 receptors (GPR32 and ALX/FPR2) in AT-RvD1 induced effects on VSMC migration and PKA activity.ResultsTreatment with AT-RvD1 induced a significant increase in cAMP levels and PKA activity in VSMCs at 5 minutes and 30 minutes, respectively. AT-RvD1 attenuated PDGF-induced VSMC migration and cytoskeletal rearrangements. These effects were attenuated by the PKA inhibitor Rp-8-Br-cAMP, suggesting cAMP/PKA involvement. Treatment of VSMC with AT-RvD1 inhibited PDGF-stimulated Rac1 activity, increased VASP phosphorylation, and attenuated paxillin localization to focal adhesions; these effects were negated by the addition of Rp-8-Br-cAMP. The effects of AT-RvD1 on VSMC migration and PKA activity were attenuated by blocking ALX/FPR2, suggesting an important role of this G-protein coupled receptor.ConclusionsOur results suggest that AT-RvD1 attenuates PDGF-induced VSMC migration via ALX/FPR2 and cAMP/PKA. Interference with Rac1, VASP and paxillin function appear to mediate the downstream effects of AT-RvD1 on VSMC migration.

Resolution of vascular injury: Specialized lipid mediators and their evolving therapeutic implications

“…Attenuation of VSMC migration by SPMs (ATL, RvD1, RvD2, RvE1, MaR1) has been a consistent finding in vitro , both with VSMC harvested from human saphenous veins (Ho, Spite et al 2010) (Miyahara, Runge et al 2013) and with arterial VSMC harvested from rodent aortas (Akagi, Chen et al 2015) (Petri, Laguna-Fernandez et al 2015, Wu, Mottola et al 2017) as well as human pulmonary arteries (Hiram, Rizcallah et al 2015). This effect has been demonstrated across several prototypic VSMC motogens including PDGF, thrombin, angiotensin II, TNF-α and IL-6 (Hiram, Rizcallah et al 2015, Mottola, Wu et al 2017, Wu, Mottola et al 2017). Associated with this effect, resolvins induce rapid and reversible changes in VSMC cell-shape with a decreased length:width ratio corresponding to an anti-migratory phenotype (Ho, Spite et al 2010, Miyahara, Runge et al 2013, Mottola, Wu et al 2017, Wu, Mottola et al 2017).…”

“…Associated with this effect, resolvins induce rapid and reversible changes in VSMC cell-shape with a decreased length:width ratio corresponding to an anti-migratory phenotype (Ho, Spite et al 2010, Miyahara, Runge et al 2013, Mottola, Wu et al 2017, Wu, Mottola et al 2017). The anti-migratory effects of AT-RvD1 in human saphenous vein VSMC appear dependent on the cAMP/PKA pathway, with downstream involvement of Rac1, VASP, and paxillin ((Mottola, Wu et al 2017)).…”

“…Attenuation of VSMC-derived ROS by SPMs has been observed in vitro after stimulation with TNF-α (RvD1, RvD2, MaR1) (Miyahara, Runge et al 2013, Chatterjee, Sharma et al 2014, Akagi, Chen et al 2015) and in vivo in rabbit femoral arteries and rat carotid arteries after angioplasty (Miyahara, Runge et al 2013, Wu, Mottola et al 2017). SPM (RvD1, MaR1) signaling in VSMC appears to involve the cAMP/PKA pathway (Chatterjee, Sharma et al 2014, Mottola, Wu et al 2017, Wu, Mottola et al 2017). …”

Perivascular delivery of resolvin D1 inhibits neointimal hyperplasia in a rabbit vein graft model

“…17,19–21,31 RvD1 exposure induces cytoskel etal changes in VSMCs corresponding to an antimigratory phenotype, 17,19,31 probably through the cyclic adenosine monophosphate-protein kinase A pathway. 38 Furthermore, SPMs have shown modest antiproliferative effects on VSMCs both in vitro 19–21,31 and in vivo. 19,20,31 …”

“…Recent work has described biosynthetic pathways of SPMs in vascular tissues, signaling mechanisms, and vascular cell-specific responses to various SPMs. 34,38,41 Definition of a “resolution index” biomarker after acute vascular injury, similar to that employed in other models of sterile inflammation (eg, peritonitis), could also be useful as a surrogate end point for translational studies. 13,42,43 Along these lines, we observed an increase in the ratio of RvD1 to the proinflammatory lipid mediator LTB 4 in vein grafts treated with RvD1-loaded films.…”