Soluble guanylyl cyclase-activated cyclic GMP-dependent protein kinase inhibits arterial smooth muscle cell migration independent of VASP-serine 239 phosphorylation

Abstract

Coronary artery disease (CAD) accounts for over half of all cardiovascular disease-related deaths. Uncontrolled arterial smooth muscle (ASM) cell migration is a major component of CAD pathogenesis and efforts aimed at attenuating its progression are clinically essential. Cyclic nucleotide signaling has long been studied for its growth-mitigating properties in the setting of CAD and other vascular disorders. Heme-containing soluble guanylyl cyclase (sGC) synthesizes cyclic guanosine monophosphate (cGMP) and maintains vascular homeostasis predominantly through cGMP-dependent protein kinase (PKG) signaling. Considering that reactive oxygen species (ROS) can interfere with appropriate sGC signaling by oxidizing the cyclase heme moiety and so are associated with several CVD pathologies, the current study was designed to test the hypothesis that heme-independent sGC activation by BAY60-2770 (BAY60) maintains cGMP levels despite heme oxidation and inhibits ASM cell migration through phosphorylation of the PKG target and actin-binding vasodilator-stimulated phosphoprotein (VASP). First, using the heme oxidant ODQ, cGMP content was potentiated in the presence of BAY60. Using a rat model of arterial growth, BAY60 significantly reduced neointima formation and luminal narrowing compared to vehicle (VEH)-treated controls. In rat ASM cells BAY60 significantly attenuated cell migration, reduced G:F actin, and increased PKG activity and VASP Ser239 phosphorylation (pVASP.S239) compared to VEH controls. Site-directed mutagenesis was then used to generate overexpressing full-length wild type VASP (FL-VASP/WT), VASP Ser239 phosphorylation-mimetic (FL-VASP/ 239D) and VASP Ser239 phosphorylation-resistant (FL-VASP/239A) ASM cell mutants. Surprisingly, FL-VASP/239D negated the inhibitory effects of FL-VASP/ WT and FL-VASP/239A cells on migration. Furthermore, when FL-VASP mutants were treated with BAY60, only the FL-VASP/239D group showed reduced migration compared to its VEH controls. Intriguingly, FL-VASP/239D abrogated the stimulatory effects of FL-VASP/WT and FL-VASP/239A cells on PKG activity. In turn, pharmacologic blockade of PKG in the presence of BAY60 reversed the inhibitory effect of BAY60 on naïve ASM cell migration. Taken together, we demonstrate for the first time that BAY60 inhibits ASM cell migration through cGMP/PKG/VASP signaling yet through mechanisms independent of pVASP.S239 and that FL-VASP overexpression regulates PKG activity in rat ASM cells. These findings implicate BAY60 as a potential pharmacotherapeutic agent against aberrant ASM growth disorders such as CAD and also establish a unique mechanism through which VASP controls PKG activity.

AMP-Activated Protein Kinase Inhibits Arterial Smooth Muscle Cell Proliferation in Vasodilator-Stimulated Phosphoprotein-Dependent Manner

“…Fluorescence was detected and analyzed using Li-Cor Odyssey Infrared Imaging System and software. Additionally, ECL Western blots were performed on cell lysates to verify In-Cell Western data for select experiments as previously performed [9][10][11]13,[20][21][22].…”

“…Expression analyses: Cells were seeded in 96-well plates until confluent and treated with vehicle (DMSO) or AICAR (1 mM in DMSO), and after specified times total and phosphorylated protein expression was determined by ECL-based Western blotting on cell homogenates and by In-Cell Western blotting on intact, adherent cells as we have described [9][10][11]13,[20][21][22]. In brief, fixed cells were permeabilized with 0.1% Triton-X, blocked with IRDye blocking buffer and treated with rabbit anti-rat primary antibodies for 1 hour at room temperature (RT).…”

“…Cell culture: Rat A7r5 ASM cells (ATCC, Manassas, VA) were cultured under sterile conditions in Dulbecco's Modified Eagles Medium (DMEM) supplemented with fetal bovine serum (FBS, 0.2-10%), 2 mM L-glutamine, a 1:500 dilution of 50 µg/mL Primocin (Invivogen) at 37°C in 95% air/5% CO 2 and were serially split and used through passage 6 as described [11,13,22].…”

“…In complement, we recently showed that AMPK has capacity to communicate with the cyclic AMP/protein kinase A (PKA) pathway in growth suppression of ASM under both in vitro and in vivo conditions [9][10][11][12][13].…”

“…Actin-binding vasodilator-stimulated phosphoprotein (VASP) is a key cytoskeletal regulator that promotes actin polymerization by delivering monomeric globular (G) actin to the barbed end of growing F-actin [14][15][16]. VASP has been implicated in the control of intra-and extra-cellular signaling events associated with transcriptional activity as well as cell migration and proliferation [13][14][15][16][17][18][19]. Due to its role as a mediator of signal transduction, differential VASP phosphorylation has also been used as a readout of discrete protein kinase signaling [17,[20][21][22], and we [9,10] and others [23] have documented capacity of AMPK to phosphorylate VASP at discrete Ser and/or Thr residues.…”