Soluble guanylyl cyclase-activated cyclic GMP-dependent protein kinase inhibits arterial smooth muscle cell migration independent of VASP-serine 239 phosphorylation.
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 BAY 60-2770 (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.
Department of Physiology, Brody School of Medicine, East Carolina University, Greenville, NC, United States.
Andrew W
AW
Holt
Department of Physiology, Brody School of Medicine, East Carolina University, Greenville, NC, United States.
Danielle N
DN
Martin
Department of Biochemistry, Brody School of Medicine, East Carolina University, Greenville, NC, United States.
Patti R
PR
Shaver
Department of Physiology, Brody School of Medicine, East Carolina University, Greenville, NC, United States.
Shaquria P
SP
Adderley
Department of Physiology, Brody School of Medicine, East Carolina University, Greenville, NC, United States.
Joshua D
JD
Stone
Department of Physiology, Brody School of Medicine, East Carolina University, Greenville, NC, United States.
Chintamani N
CN
Joshi
Department of Physiology, Brody School of Medicine, East Carolina University, Greenville, NC, United States.
Jake T
JT
Francisco
Department of Physiology, Brody School of Medicine, East Carolina University, Greenville, NC, United States.
Robert M
RM
Lust
Department of Microbiology and Immunology, Brody School of Medicine, East Carolina University, Greenville, NC, United States.
Douglas A
DA
Weidner
Department of Biochemistry, Brody School of Medicine, East Carolina University, Greenville, NC, United States.
Brian M
BM
Shewchuk
Department of Physiology, Brody School of Medicine, East Carolina University, Greenville, NC, United States. Electronic address: tulisd@ecu.edu.
David A
DA
Tulis
D000199:Actins / Q000378:metabolism; D000818:Animals; D001158:Arteries / Q000166:cytology*; D001565:Benzoates / Q000494:pharmacology; D001713:Biphenyl Compounds / Q000494:pharmacology; D015815:Cell Adhesion Molecules / Q000378:metabolism*; D002465:Cell Movement / Q000187:drug effects; D017869:Cyclic GMP-Dependent Protein Kinases / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D006845:Hydrocarbons, Fluorinated / Q000494:pharmacology; D008297:Male; D008840:Microfilament Proteins / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000378:metabolism; D032389:Myocytes, Smooth Muscle / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology; D010084:Oxidation-Reduction; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D010768:Phosphoserine; D017207:Rats, Sprague-Dawley; D015203:Reproducibility of Results; D000071756:Soluble Guanylyl Cyclase / Q000378:metabolism*; D066253:Vascular Remodeling / Q000187:drug effects; D000098662:Vasodilator-Stimulated Phosphoprotein
C528797:4-(((4-carboxybutyl) (2- (5-fluoro-2-((4'-(trifluoromethyl) biphenyl-4-yl)methoxy)phenyl)ethyl) amino)methyl)benzoic acid; D000199:Actins; D001565:Benzoates; D001713:Biphenyl Compounds; D015815:Cell Adhesion Molecules; D006845:Hydrocarbons, Fluorinated; D008840:Microfilament Proteins; D050505:Mutant Proteins; D010750:Phosphoproteins; D000098662:Vasodilator-Stimulated Phosphoprotein; D010768:Phosphoserine; D017869:Cyclic GMP-Dependent Protein Kinases; D000071756:Soluble Guanylyl Cyclase
BAY 60-2770; Migration; PKG; Smooth muscle cell; VASP; cGMP