RESUMEN
Vascular smooth muscle (VSM) proliferation and migration are key components in vessel remodeling. Cyclic nucleotide signaling is protective and has long-served as a therapeutic target against undesired VSM growth. The present work analyzed the effects of the soluble guanylate cyclase (sGC) stimulator 3-(4-amino-5-cyclopropylpyrimidine-2-yl)-1-(2-fluorobenzyl)-1H-pyrazolo[3,4-b]pyridine [BAY 41-2272 (BAY)] on VSM growth, and we hypothesize that BAY has the capacity to reduce proliferation and migration via cyclic nucleotide-driven kinase signaling. Perivascular BAY postballoon injury reduced neointimal growth by â¼ 40% compared with vehicle controls after 2 weeks. In VSM cells, BAY (10 µM) reduced proliferation by â¼ 40% after 72 h and migration by â¼ 40% after 6 h and â¼ 60% after 18 h without deleterious effects on cell viability. cGMP content peaked (248 ×) 20 min after BAY treatment and remained elevated (140 ×) through 60 min; however, BAY did not affect cAMP levels compared with controls. Conventional and In-Cell Western analyses showed increases in vasodilator-stimulated phosphoprotein (VASP) phosphorylation (pVASP) at serines 239 (3 ×) and 157 (2 ×), respective markers of cGMP- and cAMP-directed protein kinases (PKG and PKA, respectively). The PKG inhibitor YGRKKRRQRRRPPLRKKKKKH peptide (DT-2) completely reversed BAY-mediated increases in pVASPSer(239) and BAY-mediated inhibition of migration. In comparison, the PKA inhibitor peptide PKI further potentiated BAY-stimulated pVASPSer(157) and pVASPSer(239) and partially reversed the antiproliferative effects of BAY. This is the first report demonstrating the effectiveness of BAY in reducing neointimal growth with direct evidence for PKG-specific antimigratory and PKA-specific antiproliferative mechanisms. Conclusively, the sGC stimulator BAY reduces VSM growth through cGMP-dependent PKG and PKA processes, providing support for continued evaluation of its clinical utility.
Asunto(s)
Proliferación Celular/efectos de los fármacos , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de GMP Cíclico/metabolismo , Inhibidores de Crecimiento/farmacología , Músculo Liso Vascular/efectos de los fármacos , Pirazoles/farmacología , Piridinas/farmacología , Receptores Citoplasmáticos y Nucleares/agonistas , Administración Tópica , Animales , Traumatismos de las Arterias Carótidas/tratamiento farmacológico , Traumatismos de las Arterias Carótidas/patología , Ciclo Celular/efectos de los fármacos , Ensayos de Migración Celular , Supervivencia Celular/efectos de los fármacos , AMP Cíclico/análisis , GMP Cíclico/análisis , Inhibidores de Crecimiento/administración & dosificación , Inhibidores de Crecimiento/uso terapéutico , Guanilato Ciclasa , Masculino , Terapia Molecular Dirigida , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/fisiología , Fosforilación/efectos de los fármacos , Pirazoles/administración & dosificación , Pirazoles/uso terapéutico , Piridinas/administración & dosificación , Piridinas/uso terapéutico , Ratas , Ratas Sprague-Dawley , Transducción de Señal/efectos de los fármacos , Guanilil Ciclasa SolubleRESUMEN
Vascular smooth muscle (VSM) growth is integral in the pathophysiology of blood vessel diseases, and identifying approaches that have capacity to regulate VSM growth is critically essential. Cyclic nucleotide signaling has been generally considered protective in cardiac and vascular tissues and has been the target of numerous basic science and clinical studies. In this project, the influence of BAY 41-2272 (BAY), a recently described soluble guanylate cyclase stimulator and inducer of cyclic guanosine monophosphate (cGMP) synthesis, on VSM cell growth was analyzed. In rat A7R5 VSM cells, BAY significantly reduced proliferation in a dose- and time-dependent fashion. BAY activated cGMP and cyclic adenosine monophosphate (cAMP) signaling evidenced through elevated cGMP and cAMP content, increased expression of cyclic nucleotide-dependent protein kinases, and differential vasodilator-stimulated phosphoprotein phosphorylation. BAY significantly elevated cyclin E expression, decreased expression of the regulatory cyclin-dependent kinases -2 and -6, increased expression of cell cycle inhibitory p21 WAF1/Cip1 and p27 Kip1, and reduced expression of phosphorylated focal adhesion kinase. These comprehensive findings provide first evidence for the antigrowth cell cycle-regulatory properties of the neoteric agent, BAY 41-2272, in VSM and lend support for its continued study in the clinical and basic cardiovascular sciences.
Asunto(s)
Proliferación Celular/efectos de los fármacos , Inhibidores de Crecimiento/farmacología , Músculo Liso Vascular/efectos de los fármacos , Pirazoles/farmacología , Piridinas/farmacología , Animales , Ciclo Celular/efectos de los fármacos , Células Cultivadas , AMP Cíclico/fisiología , GMP Cíclico/fisiología , Ciclina E/metabolismo , Quinasa 2 Dependiente de la Ciclina/biosíntesis , Quinasa 6 Dependiente de la Ciclina/biosíntesis , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/biosíntesis , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/biosíntesis , Quinasas Ciclina-Dependientes/metabolismo , Relación Dosis-Respuesta a Droga , Proteína-Tirosina Quinasas de Adhesión Focal/biosíntesis , Guanilato Ciclasa , Músculo Liso Vascular/citología , Músculo Liso Vascular/metabolismo , Fosforilación , Ratas , Receptores Citoplasmáticos y Nucleares/agonistas , Transducción de Señal/efectos de los fármacos , Guanilil Ciclasa SolubleRESUMEN
The benzylindazole derivative 3-(5'-hydroxymethyl-2'-furyl)-1-benzyl indazole (YC-1) is an allosteric stimulator of soluble guanylate cyclase (sGC) that sensitizes the enzyme to the gaseous ligands carbon monoxide (CO) and nitric oxide (NO). In this study, we examined whether YC-1 also promotes the production of these gaseous monoxides by stimulating the expression of the inducible isoforms of heme oxygenase (HO-1) and NO synthase (iNOS) in vascular smooth muscle cells (SMCs). YC-1 increased HO-1 mRNA, protein, and promoter activity and potentiated cytokine-mediated expression of iNOS protein and NO synthesis by SMCs. The induction of HO-1 by YC-1 was unchanged by the sGC inhibitor, 1H-(1,2,4)oxadiazolo[4,3-alpha]quinozalin-1-one (ODQ) or by the protein kinase G inhibitors (8R,9S,11S)-(-)-2-methyl-9-methoxyl-9-methoxycarbonyl-8-methyl-2,3,9,10-tetrahydro-8,11-epoxy-1H,8H,11H-2,7b,11a-triazadibenzo(a,g)cyclocta9(cde)trinen-1-one (KT 5823) and YGRKKRRQRRRPPLRKKKKKH-amide (DT-2) and was not duplicated by 8-bromo-cGMP or the NO-independent sGC stimulator 5-cyclopropyl-2[1-(2-fluorobenzyl)-1H-pyrazolo [3,4-b] pyridine-3-yl] pyrimidin-4-ylamine (BAY 41-2272). However, the YC-1-mediated induction of HO-1 was inhibited by the phosphatidylinositol-3-kinase (PI3K) inhibitors wortmannin and 2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride (LY294002). In contrast, the enhancement of cytokine-stimulated iNOS expression and NO production by YC-1 was prevented by ODQ and the protein kinase A inhibitor (9S,10S, 12R)-2,3,9,10,11,12-hexahydro-10-hydroxy-9-methyl-1-oxo-9, 12-epoxy-1H-diindolo(1,2,3-fg:3',2',1'-kl)pyrrolo(3,4-i)(1,6)-benzodiazocine-10-carboxylic acid hexyl ester (KT 5720) and was mimicked by 8-bromo-cGMP and BAY 41-2272. In conclusion, these studies demonstrate that YC-1 stimulates the expression of HO-1 and iNOS in vascular SMCs via the PI3K and sGC-cGMP-protein kinase A pathway, respectively. The ability of YC-1 to sensitize sGC to gaseous monoxides and simultaneously stimulate their production through the induction of HO-1 and iNOS provides a potent mechanism by which the cGMP-dependent and -independent biological actions of this agent are amplified.