Vascular smooth muscle cell (VSMC) activation promotes a synthetic phenotype that underlies many vessel growth disorders. In this regard it has been suggested that the metabolic sensor adenosine 5'-monophosphate-activated protein kinase (AMPK) has significant antigrowth and antimetastatic properties and may serve as a viable therapeutic target. In the current study we hypothesized that AMPK reduces neointima formation following balloon injury and that this occurs through reduction in VSMC proliferation and migration. Data reveal that local or systemic dosing with the AMPK agonist 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR) significantly increased AMPK activity in vivo and inhibited neointima formation in rat carotid arteries 2 wk after injury. In primary VSMCs, AICAR inhibited migration and induced cytostatic growth arrest through increased protein phosphatase 2A-mediated inhibition of mitosis-promoting cyclin B. AICAR also significantly enhanced AMPK-specific T278 phosphorylation of the actin anticapping vasodilator-activated serum phosphoprotein, increased G- to F-actin ratios and stress fiber formation, and abrogated PDGF-stimulated S397 autophosphorylation of focal adhesion kinase, promigratory cytoplasmic accumulation of paxillin, and extracellular matrix proteolysis by matrix metalloproteinase-9. Together, these results provide compelling evidence that AMPK serves to inhibit vascular smooth muscle migration and proliferation through regulation of cytoskeletal/focal adhesion/ECM stability, increasing our knowledge of this important metabolic regulator and providing support for its continued investigation in the treatment of vascular growth disorders.
Abnormal vascular smooth muscle (VSM) growth is central in the pathophysiology of vascular disease yet fully effective therapies to curb this growth are lacking. Recent findings from our lab and others support growth control of VSM by adenosine monophosphate (AMP)-based approaches including the metabolic sensor AMP-activated protein kinase (AMPK) and cAMP-dependent protein kinase (PKA). Molecular crosstalk between AMPK and PKA has been previously suggested, yet the extent to which this occurs and its biological significance in VSM remain unclear. Considering their common AMP backbone and similar signaling characteristics, we hypothesized that crosstalk exists between AMPK and PKA in the regulation of VSM growth. Using rat primary VSM cells (VSMC), the AMPK agonist AICAR increased AMPK activity and phosphorylation of the catalytic Thr172 site on AMPK. Interestingly, AICAR also phosphorylated a suspected PKA-inhibitory Ser485 site on AMPK, and these cumulative events were reversed by the PKA inhibitor PKI suggesting possible PKA-mediated regulation of AMPK. AICAR also increased PKA activity in a reversible fashion. The cAMP stimulator forskolin increased PKA activity and completely ameliorated Ser/Thr protein phosphatase-2C activity, suggesting a potential mechanism of AMPK modulation by PKA since inhibition of PKA by PKI reduced AMPK activity. Functionally, AMPK inhibited serum-stimulated cell cycle progression and cellular proliferation; however, PKA failed to do so. Moreover, AMPK and PKA reduced PDGF-β-stimulated VSMC migration. Collectively, these results show that AMPK is capable of reducing VSM growth in both anti-proliferative and anti-migratory fashion. Furthermore, these data suggest that AMPK may be modulated by PKA and that positive feedback may exist between these two systems. These findings reveal a discrete nexus between AMPK and PKA in VSM and provide basis for metabolically-directed targets in reducing pathologic VSM growth.
Aberrant vascular smooth muscle cell (VSMC) growth is pivotal in the pathophysiology of vessel disease. We previously reported that the cyclic AMP/PK‐A system has capacity to attenuate growth of serum‐stimulated rat A7R5 VSMCs. Considering the common utilization of a 5′‐AMP substrate, in the current study we hypothesize that regulatory crosstalk occurs between adenosine monophosphate‐activated protein kinase (AMPK) and the cyclic AMP/PK‐A signaling cascades in VSM. Incubation of rat primary VSMCs with the AMPK activator AICAR significantly increased phosphorylation of both a catalytic Thr172 site and a proposed inhibitory Ser485 residue on AMPK. Concomitantly, we observed increases in AMPK activity shown by both phosphorylation of the downstream target Acetyl CoA Carboxylase (ACC) at Ser80 and an AMPK activity assay. These events were completely abridged by both the AMPK and PKA inhibitors Compound C (CC) and PKI, respectively. AICAR also induced CC‐ and PKI‐inhibitable PK‐A activation measured by vasodilator‐stimulated phosphoprotein (VASP) phosphorylation at Thr157 and a PK‐A activity assay. Lastly, preliminary flow cytometry results suggest that AMPK activity inhibits cell cycle progression from G0/G1. These provocative findings reveal a discrete nexus between AMPK and cyclic AMP/PK‐A in VSMCs and lend credence for a potential metabolic target for quelling abnormal VSMC growth.
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