formation induced by balloon injury in vivo 8-Chloro-cAMP inhibits smooth muscle cell proliferation in vitro and neointima (original) (raw)
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Journal of the American College of Cardiology, 2000
The aims of the present study were to assess 1) the effect of 8-Cl-cAMP (cyclic-3Ј-5Јadenosine monophosphate) on vascular smooth muscle cell (VSMC) proliferation in vitro and 2) the efficacy of systemic administration of 8-Cl-cAMP on neointimal formation after balloon injury in vivo. BACKGROUND Neointimal formation after vascular injury is responsible for restenosis after arterial stenting. Recently, 8-Cl-cAMP, a cAMP analogue that induces growth arrest, has been safely administered in phase I studies in humans. METHODS The effect of 8-Cl-cAMP on cell proliferation was first assessed on SMCs in vitro. To study the effects of cAMP in vivo, balloon injury was performed in 67 rats using a 2F Fogarty balloon catheter. RESULTS The 8-Cl-cAMP markedly inhibited VSMC proliferation in vitro, reduced protein kinase A (PKA) RI ␣ subunit expression, and induced PKA RII  subunit expression. In addition, 8-Cl-cAMP reduced, in a dose-dependent manner, neointimal area and neointima/media ratio after balloon injury. The proliferative activity, assessed by proliferating nuclear cell antigen immunostaining, revealed a reduction of proliferative activity of VSMCs in vivo in the 8-Cl-cAMP group. Moreover, the systemic administration of 8-Cl-cAMP did not affect renal function, blood pressure and heart rate. CONCLUSIONS We conclude that 8-Cl-cAMP potently inhibits VSMC proliferation in vitro and reduces neointima formation by balloon injury in vivo after systemic administration. These data may have a clinical relevance in designing future strategies to prevent restenosis after arterial stenting and perhaps after percutaneous transluminal coronary angioplasty.
Ending Restenosis: Inhibition of Vascular Smooth Muscle Cell Proliferation by cAMP
Cells
Increased vascular smooth muscle cell (VSMC) proliferation contributes towards restenosis after angioplasty, vein graft intimal thickening and atherogenesis. The second messenger 3′ 5′ cyclic adenosine monophosphate (cAMP) plays an important role in maintaining VSMC quiescence in healthy vessels and repressing VSMC proliferation during resolution of vascular injury. Although the anti-mitogenic properties of cAMP in VSMC have been recognised for many years, it is only recently that we gained a detailed understanding of the underlying signalling mechanisms. Stimuli that elevate cAMP in VSMC inhibit G1-S phase cell cycle progression by inhibiting expression of cyclins and preventing S-Phase Kinase Associated Protein-2 (Skp2-mediated degradation of cyclin-dependent kinase inhibitors. Early studies implicated inhibition of MAPK signalling, although this does not fully explain the anti-mitogenic effects of cAMP. The cAMP effectors, Protein Kinase A (PKA) and Exchange Protein Activated by ...
cAMP inhibits TGFβ1-induced in vitro angiogenesis
FEBS Letters, 2004
Transforming growth factor-b (TGFb1) is a proangiogenic factor both, in vitro and in vivo, that is mainly involved in the later phases of angiogenesis. In an attempt to identify genes that participate in this effect, we found that TGFb1 downregulates expression of adenylate cyclase VI. In addition, cAMP analogs (8-Bromo-cAMP) and forskolin (an adenylate cyclase activator) also reduced TGFb1-induced in vitro angiogenesis in mouse endothelial cell lines and in primary cultures of human umbilical vein endothelial cells on collagen gels. Induction of Ets-1 and plasminogen activator inhibitor-1 (PAI-1) by TGFb1 was blocked by these cAMP agonists and activators, in the absence of effects on endothelial cell viability. Moreover, the signal transduction pathways stimulated by TGFb1 were unaffected. Thus, Smad2 was normally phosphorylated and translocated to the nucleus in the presence of forskolin. In contrast, transfection studies using the PAI-1-promoter indicated that these cAMP analogues inhibit transcriptional stimulation by TGFb1. Electrophoretic mobility shift assay showed that Smad2/3 were bound normally to a TGFb1-response region in the presence of the cAMP analogs. In all, these data suggest that the cAMP pathway inhibits the transcriptional activity of Smads, that could be responsible for the block of the TGFb1-induced in vitro angiogenesis caused by this second messenger.
… of Pharmacology and …, 2011
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-5cyclopropylpyrimidine-2-yl)-1-(2-fluorobenzyl)-1H-pyrazolo[3,4b]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 ϳ40% compared with vehicle controls after 2 weeks. In VSM cells, BAY (10 M) reduced proliferation by ϳ40% after 72 h and migration by ϳ40% after 6 h and ϳ60% after 18 h without deleterious effects on cell viability. cGMP content peaked (248ϫ) 20 min after BAY treatment and remained elevated (140ϫ) 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 (3ϫ) and 157 (2ϫ), 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 BAYstimulated 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 PKAspecific 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.
Circulation, 1999
Background —Although percutaneous transluminal coronary angioplasty (PTCA) is a highly effective procedure to reduce the severity of stenotic coronary atherosclerotic disease, its long-term success is significantly limited by the high rate of restenosis. Several cellular and molecular mechanisms have been implicated in the development of restenosis post-PTCA, including vascular smooth muscle cell (VSMC) activation, migration, and proliferation. Recently, our group demonstrated that rapamycin, an immunosuppressant agent with antiproliferative properties, inhibits both rat and human VSMC proliferation and migration in vitro. In the present study, we investigated (1) whether rapamycin administration could reduce neointimal thickening in a porcine model of restenosis post-PTCA and (2) the mechanism by which rapamycin inhibits VSMCs in vivo. Methods and Results —PTCA was performed on a porcine model at a balloon/vessel ratio of 1.7±0.2. Coronary arteries were analyzed for neointimal form...
Antirestenotic Effects of a Locally Delivered Caspase Inhibitor in a Balloon Injury Model
Circulation, 2003
The precise role of arterial barotrauma-mediated apoptosis in causing restenosis is unclear. The purpose of this study was to determine if a link exists between angioplasty-mediated medial smooth muscle cell apoptosis and subsequent neointimal hyperplasia. Bilateral iliac artery angioplasty was performed in 25 male New Zealand White rabbits. Simultaneous with balloon injury, each artery was treated locally with either the caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp(Ome)-fluoromethylketone (ZVAD-fmk) or control. In the acute cohort that was survived to 4 hours (n=10, 7 high dose and 3 low dose), an apoptotic index was calculated using the terminal deoxynucleotidyl TUNEL method. In the intermediate cohort that was survived to 2 weeks (n=5), luminal reendothelialization was measured via CD-31 staining. In the chronic cohort that was survived to 4 weeks (n=10), neointimal area was measured. In the acute cohort, there was a 40% reduction in the apoptotic index with high-dose ZVAD-fmk (P=0.008) and a 33% reduction with low-dose ZVAD-fmk (P=0.08). At 2 weeks, there was no significant difference in the degree of luminal reendothelialization. However, at 4 weeks, there was a 33% (0.33+/-0.23 versus 0.22+/-0.20 mm2) (P<0.005) reduction in neointimal area in ZVAD-fmk-treated arteries. The local delivery of ZVAD-fmk during balloon injury inhibits smooth muscle cell apoptosis. This corresponds to a significant reduction in neointimal proliferation seen at 4 weeks without a significant change in the degree of reendothelialization at 2 weeks.
Journal of Pharmacology and Experimental Therapeutics, 2011
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-5cyclopropylpyrimidine-2-yl)-1-(2-fluorobenzyl)-1H-pyrazolo[3,4b]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 ϳ40% compared with vehicle controls after 2 weeks. In VSM cells, BAY (10 M) reduced proliferation by ϳ40% after 72 h and migration by ϳ40% after 6 h and ϳ60% after 18 h without deleterious effects on cell viability. cGMP content peaked (248ϫ) 20 min after BAY treatment and remained elevated (140ϫ) 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 (3ϫ) and 157 (2ϫ), 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 BAYstimulated 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 PKAspecific 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.
American Journal of Physiology-Heart and Circulatory Physiology, 2009
cAMP inhibits proliferation in most cell types, triggering different and sometimes opposing molecular pathways. p85α (phosphatidylinositol 3-kinase regulatory subunit) is phosphorylated by cAMP/PKA in certain cell lineages, but its effects on vascular smooth muscle cells (VSMCs) and endothelial cells (ECs) are unknown. In the present study, we evaluated 1) the role of p85α in the integration of cAMP/PKA-dependent signaling on the regulation of VSMC and EC growth in vitro; and 2) the effects of PKA-modified p85α on neointimal hyperplasia and endothelial healing after balloon injury in vivo. Plasmid constructs carrying wild-type and PKA-modified p85α were employed in VSMCs and ECs in vitro and after balloon injury in rat carotid arteries in vivo. cAMP/PKA reduced VSMC proliferation through p85α phosphorylation. Transfected PKA-activated p85α binds p21ras, reducing ERK1/2 activation and VSMC proliferation in vitro. In contrast, EC proliferation inhibition by cAMP is independent from PK...
Opposite effect of cAMP signaling in endothelial barriers of different origin
AJP: Cell Physiology, 2004
cAMP-mediated signaling mechanisms may destabilize or stabilize the endothelial barrier, depending on the origin of endothelial cells. Here, microvascular coronary [coronary endothelial cells (CEC)] and macrovascular aortic endothelial cell (AEC) monolayers with opposite responses to cAMP were analyzed. Macromolecule permeability, isometric force, activation state of contractile machinery [indicated by phosphorylation of regulatory myosin light chains (MLC), activity of MLC kinase, and MLC phosphatase], and dynamic changes of adhesion complex proteins (translocation of VE-cadherin and paxillin) were determined. cAMP signaling was stimulated by the adenosine receptor agonist 5′- N-(ethylcarboxamido)-adenosine (NECA), the β-adrenoceptor agonist isoproterenol (Iso), or by the adenylyl cyclase activator forskolin (FSK). Permeability was increased in CEC and decreased in AEC on stimulation with NECA, Iso, or FSK. The effects could be inhibited by the PKA inhibitor Rp-8-CPT-cAMPS and imit...