Cyclic AMP signalling promotes VSMC quiescence in healthy vessels and during vascular healing following injury. Cyclic AMP inhibits VSMC proliferation via mechanisms that are not fully understood. We investigated the role of PKA and Epac signalling on cAMP-induced inhibition of VSMC proliferation. cAMP-mediated growth arrest was PKA-dependent. However, selective PKA activation with 6-Benzoyl-cAMP did not inhibit VSMC proliferation, indicating a requirement for additional pathways. Epac activation using the selective cAMP analogue 8-CPT-2′-O-Me-cAMP, did not affect levels of hyperphosphorylated Retinoblastoma (Rb) protein, a marker of G1-S phase transition, or BrdU incorporation, despite activation of the Epac-effector Rap1. However, 6-Benzoyl-cAMP and 8-CPT-2′-O-Me-cAMP acted synergistically to inhibit Rb-hyperphosphorylation and BrdU incorporation, indicating that both pathways are required for growth inhibition. Consistent with this, constitutively active Epac increased Rap1 activity and synergised with 6-Benzoyl-cAMP to inhibit VSMC proliferation. PKA and Epac synergised to inhibit phosphorylation of ERK and JNK. Induction of stellate morphology, previously associated with cAMP-mediated growth arrest, was also dependent on activation of both PKA and Epac. Rap1 inhibition with Rap1GAP or siRNA silencing did not negate forskolin-induced inhibition of Rb-hyperphosphorylation, BrdU incorporation or stellate morphology. This data demonstrates for the first time that Epac synergises with PKA via a Rap1-independent mechanism to mediate cAMP-induced growth arrest in VSMC. This work highlights the role of Epac as a major player in cAMP-dependent growth arrest in VSMC.

Abstract-Antioxidants that prevent LDL from oxidation may reduce atherosclerosis. Salvia miltiorrhiza Bunge is a Chinese herb widely used for the treatment of atherosclerosis-related disorders. Salvianolic acid B (Sal B), a water-soluble polyphenolic antioxidant isolated from the roots of this plant, was found to scavenge 1,1-diphenyl-2-picrylhydrazyl radicals and inhibit LDL oxidation more effectively than probucol. In order to evaluate the antiatherogenic potential, New Zealand White rabbits were fed for 12 weeks a normal diet, a high cholesterol diet, a high cholesterol diet containing 1% probucol, or a high cholesterol diet containing a 5% water-soluble extract of S miltiorrhiza (SM). Both SM and probucol feeding reduced plasma cholesterol. LDLs from the SM-treated group were more resistant to Cu 2ϩ -induced oxidation and contained more vitamin E (21.7Ϯ2.1 nmol/mol LDL cholesterol) than did LDLs from the high cholesterol diet group (9.6Ϯ1.8 nmol/mol LDL cholesterol) (PϽ.005). Endothelial damage, determined at week 6, was reduced by 53% in the SM group (PϽ.01). SM treatment reduced the atherosclerotic area in the abdominal aorta by 56% (PϽ.005) and cholesterol deposition in the thoracic aorta by 50% (PϽ.005). The severity of atherosclerosis in the SM group was significantly reduced after adjustment by using cholesterol exposure as an index of the cholesterol-lowering effect. This study concludes that the reduction of atherosclerosis by SM relies not only on its cholesterol-lowering effect but more heavily on its antioxidant potential to prevent endothelial damage and inhibit LDL oxidative modification in hypercholesterolemic animals. (Arterioscler Thromb Vasc Biol. 1998;18:481-486.)

Abstract-Cyclic nucleotides inhibit vascular smooth muscle cell (VSMC) proliferation but the underlying molecular mechanisms are incompletely understood. We studied the role of S-phase kinase-associated protein-2 (Skp2), an F-box protein of SCF Skp2 ubiquitin ligase responsible for polyubiquitylation of and subsequent proteolysis of p27 Kip1 , a key step leading to cell cycle progression. Skp2 mRNA and protein were upregulated in mitogen-stimulated VSMCs and after balloon injury in rat carotid arteries, where the time course and location of Skp2 expression closely paralleled that of proliferating cell nuclear antigen. Skp2 small interference RNA (siRNA) reduced Skp2 expression, increased p27 Kip1 levels, and inhibited VSMC proliferation in vitro. cAMP-elevating agents prominently inhibited VSMC proliferation and Skp2 expression through inhibiting Skp2 transcription as well as decreasing Skp2 protein stability. Consistent with this, activation of protein kinase A, a downstream target of cAMP, was shown to negatively regulate focal adhesion kinase (FAK) phosphorylation and Skp2 expression. Adenovirus-mediated Skp2 expression reversed cAMP-induced p27Kip1 upregulation and rescued cAMP-related S-phase entry inhibition up to 50%. 8-Bromo-cGMP also moderately reduced Skp2 and cell proliferation when VSMCs were incubated with low serum concentration. Interestingly, we showed that 8-bromo-cGMP inhibited Skp2 expression also through activation of protein kinase A, not protein kinase G, which conversely enhanced FAK Y397 phosphorylation and Skp2 expression. After balloon injury of rat carotid arteries, local forskolin treatment significantly reduced FAK Y397 phosphorylation, Skp2 expression, VSMC proliferation, and subsequent neointimal thickening. These data demonstrate for the first time that Skp2 is an important factor in VSMC proliferation and its inhibition by cyclic nucleotides. Key Words: Skp2 Ⅲ cyclic nucleotides Ⅲ smooth muscle cell Ⅲ proliferation Ⅲ focal adhesion kinase V ascular smooth muscle cell (VSMC) proliferation contributes to intima formation after balloon injury with and without stenting, venous graft failure, transplant vasculopathy, and atherosclerosis. This motivates efforts to understand the regulation of VSMC proliferation and in particular to identify pathways that could mediate inhibition of proliferation. In healthy, uninjured vessels, VSMCs are quiescent and exhibit extremely low rates of proliferation, even in the presence of mitogenic stimuli. 1 The mechanisms responsible for overcoming VSMC quiescence and initiating VSMC proliferation in response to vessel injury include release of growth factors 2,3 and remodeling of the vascular extracellular matrix. 4 -6 This stimulates signaling pathways downstream of growth factor receptors or focal adhesions, respectively. [7][8][9] Signaling pathways related to cell-cell adhesion through cadherins may also be involved. 10 Cell-type specific antiproliferative effects of cyclic nucleotides (cAMP and cGMP) are well documented. 11 In particular, incre...

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Rac(1) activity regulates VSMC proliferation by controlling Skp2 levels. Activation of Rac(1) induced by balloon injury in vivo increases Skp2 levels, which promotes VSMC proliferation and intima formation. Inhibition of this novel pathway underlies the negative effects of cAMP on VSMC proliferation.

Our previous work showed that arsenic trioxide down-regulated Cx43 and attenuated the angiogenic potential of human late endothelial progenitor cells (EPC). However, the relation between Cx43 and angiogenic activity of the EPC remained unclear. In the study, human late EPC were treated with siRNA specific to Cx43 (Cx43siRNA). The expression profiles as well as activity of the treated cells were examined. In parallel, the angiogenic potential of human EPC treated with Cx43siRNA was evaluated using murine hind limb ischemic model. The results showed that, in the EPC treated with Cx43siRNA, the activity of migration, proliferation, and angiogenic potential were attenuated, accompanied by reduction in vascular endothelial growth factor (VEGF) expression. In hind limb ischemia mice, EPC treated with Cx43siRNA lost the therapeutic angiogenic potential. VEGF supplementation partially recovered the activity impaired by Cx43 down-regulation. In conclusion, reduced Cx43 expression per se in the EPC causes decreased expression of VEGF and impaired angiogenic potential of the cells. Prevention of Cx43 reduction is a potential target to maintain the angiogenic potential of the EPC.

Objective-We sought to clarify the response of endothelial connexins to hyperlipidemia and lipid-lowering therapy. Methods and Results-Aortic endothelial gap junctions were analyzed by en face immunoconfocal microscopy and electron microscopy in C57BL/6 mice subjected to the following regimens: (1) normal chow (NC) for 3 months (3 mo), (2) NC for 9 mo, (3) NC for 3 mo, followed by a cholesterol-enriched diet (CED) for 6 mo, (4) NC for 3 mo and CED for 6 mo, with simvastatin in the final week, and (5) (in apoprotein E [apoE]-deficient mice) NC and examined at 3 mo and 7 to 9 mo. In wild-type mice, connexin37 (Cx37) and Cx40 were markedly downregulated in the CED-fed animals compared with those fed NC (CED vs 9-mo NC, 77% reduction in Cx37 and 65% reduction in Cx40; both PϽ0.01).After simvastatin treatment, Cx40 remained depressed, but Cx37 recovered to 94% of the level found in non-cholesterol-fed animals (PϽ0.01). Electron microscopy demonstrated that gap junctions were smaller in animals fed the CED compared with those given simvastatin and with controls fed NC (PϽ0.01). Endothelial connexins were rare in the atherosclerotic plaques of apoE-deficient mice. 3,4 Studies in animal models have demonstrated that hyperlipidemia is associated with alterations to the junctions between neighboring endothelial cells, including gap junctions. 5,6 Gap junctions are cellmembrane protein channels made of connexin molecules, which belong to a multigene family. In mammals, connexin37 (Cx37), Cx40, and Cx43 are known to be variously expressed in endothelial cells of different sites within the vascular tree. 7,8 In vitro studies indicate that the properties of gap-junctional channels are determined by their connexin makeup, 9 and gene-knockout studies show that mice deficient in specific type(s) of endothelial connexins are associated with vascular functional defects or morphological changes. 10 -12 Furthermore, distinct differences in the distribution and expression between connexin types in the vascular wall have been demonstrated in processes related to atherogenesis, such as hypertension, 13,14 postinjury regeneration, 15,16 and aging, 17 raising the possibility that each connexin type might play a distinctive role. Although recent investigations of hyperlipidemia induced by different experimental approaches in animal models of different species have reported that endothelial gap junctions and connexins change in the hyperlipidemic state, 5,6 whether the change is specific to the model remained unclear. In addition, in those studies, the extent of endothelium examined was limited, and knowledge regarding the response of each endothelial connexin to lipid-lowering therapy was lacking. Conclusions-MouseIn this study, we examined the expression patterns of endothelial gap junctions and connexins in C57BL/6 mice fed a cholesterol-enriched diet (CED) and in the same strain of mice deprived of the apoprotein E (apoE) gene. In addition, the effect of statins, a class of potent, lipidlowering agents reported to decrease progressio...

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