Fig 1.
RvD1 epimers attenuate PDGF-induced migration in VSMCs.
AT-RvD1 inhibits the VSMC migratory response induced by PDGF-BB (10ng/ml) in a scratch assay within a dose range of 0.01–100nM (A, n = 3). 17S-RvD1 also showed a significant reduction in VSMC migration at the lowest nanomolar dose (0.01nM; B, n = 3). **P<0.01 vs. positive control, ***P<0.001 vs. positive control, ****P<0.0001 vs. positive control.
Fig 2.
AT-RvD1 increases cAMP levels and PKA activity in VSMCs.
AT-RvD1 (10nM) rapidly increases cAMP levels peaking at 5 minutes and returning to baseline at 15 minutes (A, n≥4). AT-RvD1 (10nM) also increases PKA activity in VSMC with peak levels seen at 30 minutes (B, n = 3). **P<0.01 vs. time 0.
Fig 3.
AT-RvD1 attenuates PDGF-induced migration in VSMCs by the cAMP/PKA pathway.
AT-RvD1 (10nM) attenuates PDGF-induced migration in VSMC in a scratch assay and in a Transwell assay. The PKA specific inhibitor (Rp-8-Br-cAMP 10μM) partially reverts the effect of AT-RvD1 on VSMC migration in the scratch assay (A & B, n = 3) and almost completely in the Transwell assay (C, n = 3).
Fig 4.
AT-RvD1 attenuates PDGF-induced cytoskeletal rearrangements in VSMCs via PKA.
VSMCs were stained using an actin-phalloidin staining (representative pictures are shown in panel A). PDGF-BB (10ng/ml) caused a significant increase of VSMC length to width ratio which reflects pro-migratory cytoskeletal rearrangements (P<0.05 vs. negative control). AT-RvD1 (10nM) reduced this effect by lowering the VSMC length to width ratio below the value observed in the negative control. The PKA specific inhibitor (Rp-8-Br-cAMP 10μM) reversed the effects of AT-RvD1. (B, n = 4). ***P<0.001 vs. positive control, ##P<0.01.
Fig 5.
AT-RvD1 attenuates PDGF-induced Rac1 activation via PKA.
PDGF-BB (10ng/ml) stimulation of VSMCs induces a rapid Rac1 activation peaking at 5–15 minutes and dropping at 30 minutes (A; n = 1). AT-RvD1 (10nM) attenuates Rac1 activation induced by PDGF-BB (10ng/ml) at 15 minutes; the effect of AT-RvD1 is abrogated by adding a PKA specific inhibitor (Rp-8-Br-cAMP 10μM)(B; n≥4).
Fig 6.
AT-RvD1 induces VASP phosphorylation via PKA.
AT-RvD1 alone induced VASP phosphorylation which peaked at 30 minutes as shown in a representative blot (A; n = 2). PDGF-BB alone did not change VASP phosphorylation significantly vs. negative control. AT-RvD1 (10nM) induced VASP phosphorylation in the presence of PDGF-BB as shown in a representative blot and quantification of three independent experiments. The induction of phosphorylation was abolished by addition of a PKA specific inhibitor (Rp-8-Br-cAMP 10μM)(B; n = 3). *P<0.05 vs. positive control, #P<0.05, ##P<0.01.
Fig 7.
AT-RvD1 prevents paxillin localization to focal adhesions in PDGF-stimulated VSMCs.
Total paxillin was visualized in VSMCs using immunofluorescence (representative pictures are shown in panel A). By quantifying the number of particles (normalized by cell number) we observed that PDGF-BB (10ng/ml) induced localization of paxillin to focal adhesions at the leading edge of migrating VSMCs, while AT-RvD1 (10nM) attenuated this effect. PKA inhibition (with Rp-8-Br-cAMP 10μM) reversed the AT-RvD1 effect significantly (B; n = 4). *P<0.05 vs. positive control, #P<0.05.
Fig 8.
AT-RvD1 effects on VSMC migration and cAMP levels are mediated by ALX/FPR2.
AT-RvD1 (10nM) reduced PDGF-BB-induced (10ng/ml) VSMC migration in a scratch assay, and blocking ALX/FPR2 with a specific antibody (2μg/ml) neutralized this effect whereas an anti-GPR32 specific antibody (2μg/ml) showed virtually no effect (A, n≥3). The AT-RvD1-induced increase in PKA activity at 30 minutes is almost completely reversed by an anti-ALX/FPR2 specific antibody (2μg/ml) whereas an anti-GPR32 specific antibody (2μg/ml) showed only a not statistically significant partial reversal (B; n = 6). ***P<0.001 vs. positive control, #P<0.05.
Fig 9.
Schematic of proposed pathway by which RvD1 modulates VSMC migration.
The schematic shows a summary of our findings in a proposed simplified mechanism for the effects of AT-RvD1 on VSMC migration. AT-RvD1 increases cAMP levels at least by activating ALX/FPR2; the subsequent activation of PKA interferes with actin polymerization by inhibiting VASP (by phosphorylation) and Rac1, and with focal adhesion formation by decreasing paxillin localization to the cell leading edge. “R” and “C” represent the regulatory and the catalytic subunits of PKA, respectively.