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DNA hypomethylation at theWe conducted multiple independent experiments to assess whether DNA hypomethylation atObservationally, DNA methylation atSmoking-induced epigenetic DNA hypomethylation at

Adverse childhood experiences (ACEs) are a well-documented HIV-risk factor, but less is known about the relationship between ACEs and different HIV testing strategies. This study used data from an LGBTQ + community health assessment, that was part of a multi-staged community-based participatory research project in San Antonio, Texas. Overall, 464 young men who have sex with men (YMSM; 36-years-old) completed an online, cross-sectional survey that included questions about ACEs and HIV testing behavior. An association between increased ACEs exposure and the odds of clinic-based testing and HIVST HIV significantly decreased relative to never testing for HIV. Additionally, greater ACEs exposure was significantly associated with increased odds of reporting community-based testing (AOR = 1.09, 95% CI = 1.00, 1.20) and significantly reduced odds of HIV self-testing (AOR = 0.72, 95% CI = 0.63, 0.82) compared to clinic-based testing. Cumulative ACEs exposure is important in understanding HIV testing behaviors in YMSM and should be considered when developing HIV testing programs.

Background: DNA hypomethylation at the F2RL3 (F2R like thrombin or trypsin receptor 3) locus has been associated with both smoking and atherosclerotic cardiovascular disease; whether these smoking-related associations form a pathway to disease is unknown. F2RL3 encodes protease-activated receptor 4, a potent thrombin receptor expressed on platelets. Given the role of thrombin in platelet activation and the role of thrombus formation in myocardial infarction, alterations to this biological pathway could be important for ischemic cardiovascular disease. Methods: We conducted multiple independent experiments to assess whether DNA hypomethylation at F2RL3 in response to smoking is associated with risk of myocardial infarction via changes to platelet reactivity. Using cohort data (N=3205), we explored the relationship between smoking, DNA hypomethylation at F2RL3 , and myocardial infarction. We compared platelet reactivity in individuals with low versus high DNA methylation at F2RL3 (N=41). We used an in vitro model to explore the biological response of F2RL3 to cigarette smoke extract. Finally, a series of reporter constructs were used to investigate how differential methylation could impact F2RL3 gene expression. Results: Observationally, DNA methylation at F2RL3 mediated an estimated 34% of the smoking effect on increased risk of myocardial infarction. An association between methylation group (low/high) and platelet reactivity was observed in response to PAR4 (protease-activated receptor 4) stimulation. In cells, cigarette smoke extract exposure was associated with a 4.9% to 9.3% reduction in DNA methylation at F2RL3 and a corresponding 1.7-(95% CI, 1.2–2.4, P =0.04) fold increase in F2RL3 mRNA. Results from reporter assays suggest the exon 2 region of F2RL3 may help control gene expression. Conclusions: Smoking-induced epigenetic DNA hypomethylation at F2RL3 appears to increase PAR4 expression with potential downstream consequences for platelet reactivity. Combined evidence here not only identifies F2RL3 DNA methylation as a possible contributory pathway from smoking to cardiovascular disease risk but from any feature potentially influencing F2RL3 regulation in a similar manner.

Cardiac fibrosis is associated with increased stiffness of the myocardial extracellular matrix (ECM) in part mediated by increased cardiac fibroblast proliferation However, our understanding of the mechanisms regulating cardiac fibroblast proliferation are incomplete. Cardiac fibrosis is associated with increased stiffness of the myocardial extracellular matrix (ECM). Here we characterise a novel mechanism involving a combined activation of YAP targets RUNX Family Transcription Factor 2 (RUNX2) and TEA Domain Transcription Factor (TEAD).We demonstrate that cardiac fibroblast proliferation is enhance by interaction with a stiff ECM compared to a soft ECM. This is associated with activation of the transcriptional co-factor, YAP. We demonstrate that this stiffness induced activation of YAP enhances the transcriptional activity of both TEAD and RUNX2 transcription factors. Inhibition of either TEAD or RUNX2, using gene silencing, expression of dominant-negative mutants or pharmacological inhibition, reduces cardiac fibroblast proliferation. Using mutants of YAP, defective in TEAD or RUNX2 activation ability, we demonstrate a dual role of YAP-mediated activation of TEAD and RUNX2 for substrate stiffness induced cardiac fibroblast proliferation.Our data highlights a previously unrecognised role of YAP mediated RUNX2 activation for cardiac fibroblast proliferation in response to increased ECM stiffness.

Ligand-induced activation of Exchange Protein Activated by cAMP-1 (EPAC1) is implicated in numerous physiological and pathological processes, including cardiac fibrosis where changes in EPAC1 expression have been detected. However, little is known about how EPAC1 expression is regulated. Therefore, we investigated regulation of EPAC1 expression by cAMP in cardiac fibroblasts.Elevation of cAMP using forskolin, cAMP-analogues or adenosine A2B-receptor activation significantly reduced EPAC1 mRNA and protein levels and inhibited formation of F-actin stress fibres. Inhibition of actin polymerisation with cytochalasin-D, latrunculin-B or the ROCK inhibitor, Y-27632, mimicked effects of cAMP on EPAC1 mRNA and protein levels. Elevated cAMP also inhibited activity of an EPAC1 promoter-reporter gene, which contained a consensus binding element for TEAD, which is a target for inhibition by cAMP. Inhibition of TEAD activity using siRNA-silencing of its co-factors YAP and TAZ, expression of dominant-negative TEAD or treatment with YAP-TEAD inhibitors, significantly inhibited EPAC1 expression. However, whereas expression of constitutively-active YAP completely reversed forskolin inhibition of EPAC1-promoter activity it did not rescue EPAC1 mRNA levels. Chromatin-immunoprecipitation detected a significant reduction in histone3-lysine27-acetylation at the EPAC1 proximal promoter in response to forskolin stimulation. HDAC1/3 inhibition partially reversed forskolin inhibition of EPAC1 expression, which was completely rescued by simultaneously expressing constitutively active YAP.Taken together, these data demonstrate that cAMP downregulates EPAC1 gene expression via disrupting the actin cytoskeleton, which inhibits YAP/TAZ-TEAD activity in concert with HDAC-mediated histone deacetylation at the EPAC1 proximal promoter. This represents a novel negative feedback mechanism controlling EPAC1 levels in response to cAMP elevation.

The Hippo pathway is an important regulator of cell growth, proliferation, and migration. TEAD transcription factors, which lie at the core of the Hippo pathway, are essential for regulation of organ growth and wound repair. Dysregulation of TEAD and its regulatory cofactor Yes-associated protein (YAP) have been implicated in numerous human cancers and hyperproliferative pathological processes. Hence, the YAP–TEAD complex is a promising therapeutic target. Here, we use in silico molecular docking using Bristol University Docking Engine to screen a library of more than 8 million druglike molecules for novel disrupters of the YAP–TEAD interaction. We report the identification of a novel compound (CPD3.1) with the ability to disrupt YAP–TEAD protein–protein interaction and inhibit TEAD activity, cell proliferation, and cell migration. The YAP–TEAD complex is a viable drug target, and CPD3.1 is a lead compound for the development of more potent TEAD inhibitors for treating cancer and other hyperproliferative pathologies.

Actin dynamics regulate cell behaviour in response to physiological signals. Here we demonstrate a novel role for nuclear actin in inhibiting cell proliferation and migration. We demonstrate that physiological signals that elevate cAMP, which is anti-mitogenic in vascular smooth muscle cells, increases nuclear actin monomer levels. Expression of a nuclear-targeted polymerisation-defective actin mutant (NLS-ActinR62D) inhibited proliferation and migration. Preventing nuclear actin monomer accumulation by enhancing its nuclear export or polymerisation reversed the anti-mitogenic and anti-migratory effects of cAMP. Transcriptomic analysis identified repression of proliferation and migration associated genes regulated by serum response factor (SRF) and TEA Domain (TEAD) transcription factors. Accordingly, NLS-ActinR62D inhibited SRF and TEAD activity and target gene expression, and these effects were reversed by constitutively-active mutants of the TEAD and SRF co-factors YAP, TAZ and MKL1. In summary, intranuclear actin inhibits proliferation and migration by inhibiting YAP-TEAD and MKL-SRF activity. This mechanism explains the anti-mitogenic and anti-migratory properties of physiological signals that elevate cAMP. Summary McNeill et al show that increased levels of intranuclear actin monomer inhibit cell proliferation and migration by inhibiting MKL1-SRF and YAP/TAZ-TEAD-dependent gene expression. This mechanism mediates the anti-mitogenic and anti-migratory effects of physiological signals that elevate cyclic-AMP., Highlights • Elevated cAMP increases levels of nuclear actin in vascular smooth muscle cells. • Nuclear actin inhibits vascular smooth muscle cell proliferation and migration. • Nuclear actin mediates anti-mitogenic and anti-migratory properties of cAMP. • TEAD and SRF inhibition mediates growth inhibitory effects of nuclear actin. • TEAD and SRF inhibition mediates inhibition of migration by nuclear actin.

Protease-activated receptor 4 (PAR4) is a potent thrombin receptor. Epigenetic control of theF2RL3locus (which encodes for PAR4) via DNA methylation is associated with both smoking and cardiovascular disease. We examined the association between DNA hypomethylation atF2RL3and risk of cardiovascular disease, focusing on acute myocardial infarction (AMI) (n=853 cases / 2,352 controls). We usedin vitrocell models to dissect the role of DNA methylation in regulating expression ofF2RL3.We investigated the interplay betweenF2RL3DNA methylation and platelet function in human (n=41). Lastly, we used Mendelian randomization to unify observational and functional work by assessing evidence for causal relationships using data from UK Biobank (n=407,141) and CARDIoGRAMplusC4D (n=184,305). Observationally, one standard deviation (SD) decrease in DNA methylation atF2RL3was associated with a 25% increase in the odds of AMI.In vitro, short-term exposure of cells to cigarette smoke reducedF2RL3DNA methylation and increased gene expression. Transcriptional assays flagged a role for a CEBP recognition sequence in modulating the enhancer activity ofF2RL3exon 2. Lower DNA methylation atF2RL3was associated with increased platelet reactivity in human. The estimated casual odds ratio of ischaemic heart disease was 1.03 (95% CI: 1.00, 1.07) per 1 SD decrease inF2RL3DNA. In conclusion, we show that DNA methylation-dependent platelet activation is part of a complex system of features contributing to cardiovascular health. Tailoring therapeutic intervention to new knowledge ofF2RL3/PAR4 function should be explored to ameliorate the detrimental effects of this risk factor on cardiovascular health.One sentence summaryDNA methylation-dependent platelet activation is a likely causal contributor to cardiovascular health.

Hypoxia is a hallmark of solid tumours and a key physiological feature distinguishing cancer from normal tissue. However, a major challenge remains in identifying tractable molecular targets that hypoxic cancer cells depend on for survival. Here, we used SILAC‐based proteomics to identify the orphan G protein‐coupled receptor GPRC5A as a novel hypoxia‐induced protein that functions to protect cancer cells from apoptosis during oxygen deprivation. Using genetic approaches in vitro and in vivo, we reveal HIFs as direct activators of GPRC5A transcription. Furthermore, we find that GPRC5A is upregulated in the colonic epithelium of patients with mesenteric ischaemia, and in colorectal cancers high GPRC5A correlates with hypoxia gene signatures and poor clinical outcomes. Mechanistically, we show that GPRC5A enables hypoxic cell survival by activating the Hippo pathway effector YAP and its anti‐apoptotic target gene BCL2L1. Importantly, we show that the apoptosis induced by GPRC5A depletion in hypoxia can be rescued by constitutively active YAP. Our study identifies a novel HIF‐GPRC5A‐YAP axis as a critical mediator of the hypoxia‐induced adaptive response and a potential target for cancer therapy.

Vascular smooth muscle cell (VSMC) proliferation has been implicated in the development of restenosis after angioplasty, vein graft intimal thickening and atherogenesis. We investigated the mechanisms underlying positive and negative regulation of VSMC proliferation by the transcription factor cyclic AMP response element binding protein (CREB). Incubation with the cAMP elevating stimuli, adenosine, prostacyclin mimetics or low levels of forksolin activated CREB without changing CREB phosphorylation on serine-133 but induced nuclear translocation of the CREB co-factors CRTC-2 and CRTC-3. Overexpression of CRTC-2 or -3 significantly increased CREB activity and inhibited VSMC proliferation, whereas CRTC-2/3 silencing inhibited CREB activity and reversed the anti-mitogenic effects of adenosine A2B receptor agonists. By contrast, stimulation with serum or PDGFBB significantly increased CREB activity, dependent on increased CREB phosphorylation at serine-133 but not on CRTC-2/3 activation. CREB silencing significantly inhibited basal and PDGF induced proliferation. These data demonstrate that cAMP activation of CREB, which is CRTC2/3 dependent and serine-133 independent, is anti-mitogenic. Growth factor activation of CREB, which is serine-133-dependent and CRTC2/3 independent, is pro-mitogenic. Hence, CREB plays a dual role in the regulation of VSMC proliferation with the mode of activation determining its pro- or anti-mitogenic function.

Proliferation and migration of vascular smooth muscle cells (VSMCs) or endothelial cell (ECs) promote or inhibit, respectively, restenosis after angioplasty, vein graft intimal thickening and atherogenesis. Here we investigated the effects of cAMP-induced cytoskeletal remodelling on the serum response factor (SRF) co-factors Megakaryoblastic Leukemia-1 and -2 (MKL1 and MKL2) and their role in controlling VSMC and EC proliferation and migration. Elevation of cAMP using forskolin, dibutyryl-cAMP (db-cAMP), BAY60-6583 or Cicaprost induced rapid cytoskeleton remodelling and inhibited proliferation and migration in VSMCs but not EC. Furthermore, elevated cAMP inhibited mitogen-induced nuclear-translocation of MKL1 and MKL2 in VSMCs but not ECs. Forskolin also significantly inhibited serum response factor (SRF)-dependent reporter gene (SRE-LUC) activity and mRNA expression of pro-proliferative and pro-migratory MKL1/2 target genes in VSMCs but not in ECs. In ECs, MKL1 was constitutively nuclear and MKL2 cytoplasmic, irrespective of mitogens or cAMP. Pharmacological or siRNA inhibition of MKL1 significantly inhibited the proliferation and migration of VSMC and EC. Our new data identifies and important contribution of MKL1/2 to explaining the strikingly different response of VSMCs and ECs to cAMP elevation. Elucidation of these pathways promises to identify targets for specific inhibition of VSMC migration and proliferation.

Research studies in psychology and education often seek to detect changes or growth in an outcome over a duration of time. This research provides a solution to those interested in estimating latent traits from psychological measures that rely on human raters. Rater effects potentially degrade the quality of scores in constructed response and performance assessments. We develop an extension of the hierarchical rater model (HRM), which yields estimates of latent traits that have been corrected for individual rater bias and variability, for ratings that come from longitudinal designs. The parameterization, called the longitudinal HRM (L-HRM), includes an autoregressive time series process to permit serial dependence between latent traits at adjacent timepoints, as well as a parameter for overall growth. We evaluate and demonstrate the feasibility and performance of the L-HRM using simulation studies. Parameter recovery results reveal predictable amounts and patterns of bias and error for most parameters across conditions. An application to ratings from a study of character strength demonstrates the model. We discuss limitations and future research directions to improve the L-HRM.

Aims Cyclic AMP inhibits vascular smooth muscle cell (VSMC) proliferation which is important in the aetiology of numerous vascular diseases. The anti-mitogenic properties of cAMP in VSMC are dependent on activation of protein kinase A (PKA) and exchange protein activated by cAMP (EPAC), but the mechanisms are unclear. Methods and results Selective agonists of PKA and EPAC synergistically inhibited Egr1 expression, which was essential for VSMC proliferation. Forskolin, adenosine, A2B receptor agonist BAY60-6583 and Cicaprost also inhibited Egr1 expression in VSMC but not in endothelial cells. Inhibition of Egr1 by cAMP was independent of cAMP response element binding protein (CREB) activity but dependent on inhibition of serum response element (SRE) activity. SRF binding to the Egr1 promoter was not modulated by cAMP stimulation. However, Egr1 expression was dependent on the SRF co-factors Elk1 and 4 but independent of MAL. Inhibition of SRE-dependent Egr1 expression was due to synergistic inhibition of Rac1 activity by PKA and EPAC, resulting in rapid cytoskeleton remodelling and nuclear export of ERK1/2. This was associated with de-phosphorylation of the SRF co-factor Elk1. Conclusion cAMP inhibits VSMC proliferation by rapidly inhibiting Egr1 expression. This occurs, at least in part, via inhibition of Rac1 activity leading to rapid actin-cytoskeleton remodelling, nuclear export of ERK1/2, impaired Elk1-phosphorylation and inhibition of SRE activity. This identifies one of the earliest mechanisms underlying the anti-mitogenic effects of cAMP in VSMC but not in endothelial cells, making it an attractive target for selective inhibition of VSMC proliferation., Highlights • cAMP inhibits VSMC proliferation by rapidly inhibiting Egr1 expression. • PKA and Epac synergise to inhibit Egr1 expression. • cAMP-mediated inhibition of Egr1 is cell-type specific. • cAMP inhibits SRE-dependent Egr1 transcription. • cAMP inhibits Egr1 via nuclear export of ERK1/2 and de-phosphorylation of Elk1.

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 (EPAC) act together to inhibit VSMC proliferation by inducing Cyclic-AMP Response Element Binding protein (CREB) activity and inhibiting members of the RhoGTPases, which results in remodelling of the actin cytoskeleton. Cyclic-AMP induced actin remodelling controls proliferation by modulating the activity of Serum Response Factor (SRF) and TEA Domain Transcription Factors (TEAD), which regulate expression of genes required for proliferation. Here we review recent research characterising these mechanisms, highlighting novel drug targets that may allow the anti-mitogenic properties of cAMP to be harnessed therapeutically to limit restenosis.

Phenomenological study findings identified police social media site use practices that law enforcement leadership may wish to consider incorporating. Findings showed that members of the public in their lived experiences desired two-way asynchronous and synchronous interpersonal communication capabilities when accessing social media platforms maintained by law enforcement agencies. Improved police social media use practices in these areas may be a gateway to building stronger community relations and using social media as an instrument to aid in crime solving. The themes identified included crime solving, building community-relations, asynchronous versus synchronous engagement, humanistic branding, and public image.

Endothelial dysfunction caused by the combined action of disturbed flow, inflammatory mediators and oxidants derived from cigarette smoke is known to promote coronary atherosclerosis and increase the likelihood of myocardial infarctions and strokes. Conversely, laminar flow protects against endothelial dysfunction, at least in the initial phases of atherogenesis. We studied the effects of TNFα and cigarette smoke extract on human coronary artery endothelial cells under oscillatory, normal laminar and elevated laminar shear stress for a period of 72 hours. We found, firstly, that laminar flow fails to overcome the inflammatory effects of TNFα under these conditions but that cigarette smoke induces an anti-oxidant response that appears to reduce endothelial inflammation. Elevated laminar flow, TNFα and cigarette smoke extract synergise to induce expression of the transcriptional regulator activating transcription factor 3 (ATF3), which we show by adenovirus driven overexpression, decreases inflammatory gene expression independently of activation of nuclear factor-κB. Our results illustrate the importance of studying endothelial dysfunction in vitro over prolonged periods. They also identify ATF3 as an important protective factor against endothelial dysfunction. Modulation of ATF3 expression may represent a novel approach to modulate proinflammatory gene expression and open new therapeutic avenues to treat proinflammatory diseases.

Background Interferon-γ (IFN-γ) or interleukin-4 (IL-4) drives widely different transcriptional programs in macrophages. However, how IFN-γ and IL-4 alter expression of histone-modifying enzymes involved in epigenetic regulation and how this affects the resulting phenotypic polarization is incompletely understood. Methods and results We investigated steady-state messenger RNA levels of 84 histone-modifying enzymes and related regulators in colony-stimulating factor-1 differentiated primary human macrophages using quantitative polymerase chain reaction. IFN-γ or IL-4 treatment for 6–48 h changed 11 mRNAs significantly. IFN-γ increased CIITA, KDM6B, and NCOA1, and IL-4 also increased KDM6B by 6 h. However, either cytokine decreased AURKB, ESCO2, SETD6, SUV39H1, and WHSC1, whereas IFN-γ alone decreased KAT2A, PRMT7, and SMYD3 mRNAs only after 18 h, which coincided with decreased cell proliferation. Rendering macrophages quiescent by growth factor starvation or adenovirus-mediated overexpression of p27kip1 inhibited expression of AURKB, ESCO2, SUV39H1, and WHSC1, and mRNA levels were restored by overexpressing the S-phase transcription factor E2F1, implying their expression, at least partly, depended on proliferation. However, CIITA, KDM6B, NCOA1, KAT2A, PRMT7, SETD6, and SMYD3 were regulated independently of effects on proliferation. Silencing KDM6B, the only transcriptional activator upregulated by both IFN-γ and IL-4, pharmacologically or with short hairpin RNA, blunted a subset of responses to each cytokine. Conclusion These findings demonstrate that IFN-γ or IL-4 can regulate the expression of histone acetyl transferases and histone methyl transferases independently of effects on proliferation and that upregulation of the histone demethylase, KDM6B, assists phenotypic polarization by both cytokines.

Background and Purpose Myocardial cAMP elevation confers cardioprotection against ischaemia/reperfusion (I/R) injury. cAMP activates two independent signalling pathways, PKA and Epac. This study investigated the cardiac effects of activating PKA and/or Epac and their involvement in cardioprotection against I/R. Experimental Approach Hearts from male rats were used either for determination of PKA and PKC activation or perfused in the Langendorff mode for either cardiomyocyte isolation or used to monitor functional activity at basal levels and after 30 min global ischaemia and 2 h reperfusion. Functional recovery and myocardial injury during reperfusion (LDH release and infarct size) were evaluated. Activation of PKA and/or Epac in perfused hearts was induced using cell permeable cAMP analogues in the presence or absence of inhibitors of PKA, Epac and PKC. H9C2 cells and cardiomyocytes were used to assess activation of Epac and effect on Ca2+ transients. Key Results Selective activation of either PKA or Epac was found to trigger a positive inotropic effect, which was considerably enhanced when both pathways were simultaneously activated. Only combined activation of PKA and Epac induced marked cardioprotection against I/R injury. This was accompanied by PKCε activation and repressed by inhibitors of PKA, Epac or PKC. Conclusion and Implications Simultaneous activation of both PKA and Epac induces an additive inotropic effect and confers optimal and marked cardioprotection against I/R injury. The latter effect is mediated by PKCε activation. This work has introduced a new therapeutic approach and targets to protect the heart against cardiac insults.

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., Research Highlights ► PKA is required but not sufficient for the anti-mitogenic effects of cAMP in VSMC. ► cAMP-induced smooth muscle cell growth arrest is Epac-dependent but Rap1-independent. ► PKA and Epac synergise to inhibit smooth muscle cell proliferation. ► cAMP inhibits ERK and JNK phosphorylation in a PKA- and Epac-dependent manner. ► cAMP-induced stellate morphology of SMC requires PKA and Epac.

SUMMARY The clinical benefits of percutaneous coronary intervention, the most prevalent procedure nowadays for the treat- ment of symptomatic coronary artery disease, are frequently offset by the occurrence of vascular restenosis. Al- though the introduction of drug-eluting stents has significantly reduced restenotic rates, the rare, but potentially fatal, delayed thrombosis remains a clinical threat. Further refinement of the drug-eluting stent based on a better understanding of cell cycle regulation between the vascular smooth muscle cell (VSMC) and endothelial cell (EC) is required. In this review, we discuss the role of S-phase kinase-associated protein-2 (Skp2), previously known as an oncoprotein, in the regulation of VSMC proliferation and its signaling axis. The currently available evidence sug- gests that the Rac1-Skp2-p27 Kip1 signaling axis acts as a common final pathway for many factors that regulate VSMC proliferation, such as growth factors, extracellular matrices and cyclic nucleotides. Importantly, although EC prolif- eration is also shown to be regulated by the same axis, cAMP seems to regulate this axis differentially between VSMC and EC, rendering the underlying mechanism of this differential regulation a promising target for the development of a new generation of drug-eluting stent. (International Journal of Gerontology 2008; 2(4): 158-166)

Background and purpose: To determine whether there is an association between vascular NADPH oxidase (NOX), superoxide, the small GTPase Rac1 and PDE type 5 (PDE5) in human vascular smooth muscle cell (hVSMCs). Experimental approach: hVSMCs were incubated with xanthine–xanthine oxidase (X-XO; a superoxide generating system) or the thromboxane A2 analogue, U46619 (±superoxide dismutase (SOD) or apocynin) for 16 h. The expression of PDE5 and NOX-1 was assessed using Western blotting and superoxide measured. The role of Rac1 in superoxide generation was assessed by overexpressing either the dominant-negative or constitutively active Rac isoforms. The effects of iloprost, DETA-NONOate and the Rho-kinase inhibitor, Y27632, on PDE5 and NOX-1 expression were also studied. Key results: Following 16 h incubation, U46619 and X-XO promoted the expression of PDE5 and NOX-1, an effect blocked by SOD or apocynin when co-incubated over the same time course. X-XO and U46619 both promoted the formation of superoxide. Overexpression of dominant-negative Rac1 or addition of iloprost, DETA-NONOate or Y27632 completely blocked both superoxide release and PDE5 protein expression and activity. Conclusions and implications: These data demonstrate that superoxide derived from NOX upregulates the expression of PDE5 in human VSMCs. As PDE5 hydrolyses cyclic GMP, this effect may blunt the vasculoprotective actions of NO. British Journal of Pharmacology (2008) 155, 847–856; doi:10.1038/bjp.2008.300; published online 28 July 2008

Aims Vascular smooth muscle cell (VSMC) proliferation contributes to intima formation after angioplasty or venous by-pass grafting, and during atherosclerosis. VSMC proliferation requires degradation of p27Kip1 promoted by S-phase kinase-associated protein-2 (Skp2), an F-box protein component of the Skp-Cullin-F-boxSkp2 ubiquitin-ligase. We investigated the role of Rac1 in the regulation of Skp2 in rat VSMC. Methods and results Rat carotid balloon injury increased Rac1 activity. Rho GTPase inhibition with Clostridium difficile Toxin B or specific Rac1 inhibition with adenovirus-mediated expression of dominant-negative Rac1 reduced Skp2 levels, and VSMC proliferation in vitro and intima formation in vivo following carotid balloon injury. Inhibition of Skp2 expression and proliferation by dominant-negative Rac1 was reversed by exogenous Skp2. Elevation of endogenous adenosine 3′,5′-cyclic monophosphate (cAMP) with forskolin-inhibited Rac1 activity, reduced Skp2, increased p27Kip1 and inhibited VSMC proliferation, effects that were reversed by constitutively active Rac1. These effects were independent of Rac1 Cdc42/Rac interactive binding (CRIB)-domain effector proteins but associated with Rac1-dependent actin polymerization. Conclusion Rac1 activity regulates VSMC proliferation by controlling Skp2 levels. Activation of Rac1 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.

Communication technologies are often proposed to level the playing field for individuals with disabilities, but the benefits may be magnified for deaf individuals in particular due to the communication barriers experienced by these individuals. In this paper, we set out to test the assumption that increased engagement with communication technology, specifically computer-mediated communication, during adolescence would contribute to actual attainment gains in adult life for deaf individuals in three domains: life, education, and employment. A secondary analysis using the National Longitudinal Transition Study 2 (NLTS2) was conducted, allowing for a longitudinal examination of deaf individuals' experiences in the transition from adolescence to adulthood. Findings revealed that deaf individuals who engaged with computer-mediated communication at higher frequencies during adolescence did not reveal discernible gains in adult life attainments in any domain. We propose that the benefits of communication technology only go so far, and that achieving greater equitable outcomes for deaf individuals requires larger systemic change.

Aims Inhibition of vascular smooth muscle cell (VSMC) proliferation by intracellular cAMP prevents excessive neointima formation and hence angioplasty restenosis and vein-graft failure. These protective effects are mediated via actin-cytoskeleton remodelling and subsequent regulation of gene expression by mechanisms that are incompletely understood. Here we investigated the role of components of the growth-regulatory Hippo pathway, specifically the transcription factor TEAD and its co-factors YAP and TAZ in VSMC. Methods and results Elevation of cAMP using forskolin, dibutyryl-cAMP or the physiological agonists, Cicaprost or adenosine, significantly increased phosphorylation and nuclear export YAP and TAZ and inhibited TEAD-luciferase report gene activity. Similar effects were obtained by inhibiting RhoA activity with C3-transferase, its downstream kinase, ROCK, with Y27632, or actin-polymerisation with Latrunculin-B. Conversely, expression of constitutively-active RhoA reversed the inhibitory effects of forskolin on TEAD-luciferase. Forskolin significantly inhibited the mRNA expression of the pro-mitogenic genes, CCN1, CTGF, c-MYC and TGFB2 and this was reversed by expression of constitutively-active YAP or TAZ phospho-mutants. Inhibition of YAP and TAZ function with RNAi or Verteporfin significantly reduced VSMC proliferation. Furthermore, the anti-mitogenic effects of forskolin were reversed by overexpression of constitutively-active YAP or TAZ. Conclusion Taken together, these data demonstrate that cAMP-induced actin-cytoskeleton remodelling inhibits YAP/TAZ–TEAD dependent expression of pro-mitogenic genes in VSMC. This mechanism contributes novel insight into the anti-mitogenic effects of cAMP in VSMC and suggests a new target for intervention., Graphical abstract Elevated cAMP inhibits the activity of Rho GTPases and thus antagonises actin polymerisation. Impaired actin polymerisation induces the phosphorylation and nuclear export of YAP and TAZ. The nuclear export of YAP and TAZ reduces the activity of TEAD transcription factors and the transcription of TEAD-dependent cell-cycle genes. This mechanism underlies the anti-mitogenic effects of cAMP in vascular smooth muscle cells., Highlights • Elevated cAMP induces phosphorylation and nuclear export of YAP and TAZ in VSMC. • Elevated cAMP inhibits TEAD-dependent transcription of proliferation genes in VSMC. • YAP and TAZ are essential for VSMC proliferation. • The anti-mitogenic effects of cAMP in VSMC are dependent on inhibition of YAP/TAZ–TEAD mediated gene expression.

Objective: Proliferation of vascular smooth muscle cells (VSMC) is an important event in atherogenesis, in-stent restenosis and late vein-graft failure. Cell-cycle progression is positively regulated by cyclin:cdk complexes and negatively regulated by cyclin-dependent kinase inhibitors, including p21Cip1. Here we investigate the mechanisms regulating p21Cip1 levels in VSMCs and its role in controlling VSMC proliferation. Methods and results: We studied the S-phase-associated kinase protein-2 (Skp2), an F-box protein implicated in the ubiquitination of p21Cip1. Overexpression of wild-type Skp2 or dominant-negative Skp2 decreased or increased p21Cip1 levels, respectively. Interestingly, levels of endogenous p21Cip1 and Skp2 were both increased in a phosphoinositide PI 3-kinase-dependent manner in late G1 phase. Increased expression of p21Cip1 occurred despite significantly increased Skp2-mediated proteasomal degradation. To determine the role of p21Cip1 in regulating VSMC proliferation, we used adenovirus-mediated overexpression and small-interfering RNA to elevate or silence p21Cip1 expression, respectively. Overexpression of p21Cip1 significantly inhibited VSCM proliferation. p21Cip1 silencing also inhibited proliferation and increased apoptotic cell death. Conclusions: Taken together, this data demonstrates that a balance between PI 3-kinase-driven upregulation and Skp2-mediated degradation controls the level of p21Cip1, which regulates VSMC proliferation in a biphasic manner. Low levels of p21Cip1 are also essential to counter apoptosis during cell-cycle progression.

Smooth muscle cell (SMC) proliferation is suppressed in intact blood vessels but stimulated in atherosclerosis, restenosis after angioplasty, and vein graft disease. The cyclin-dependent kinase inhibitors, including p27Kip1, play important roles in maintaining SMC quiescence. Levels of p27Kip1 are dependent on attachment to and the composition of the extracellular matrix (ECM). Here we sought to elucidate mechanisms underlying the ECM-dependent regulation of p27Kip1 and hence, SMC proliferation. Serum stimulation decreased p27Kip1 levels in isolated SMC but not in rat aorta. The effect was post-translational and mediated by proteasomal degradation. We studied the S-phase-associated kinase protein-2 (Skp-2), an F-box protein involved in ubiquitination and proteasome-mediated degradation. Skp-2 protein is strongly induced by serum from undetectable levels in isolated SMCs but remains undetectable in aorta; Skp-2 mRNA is also lower in aorta. Overexpression of wild-type Skp-2 in SMCs decreased p27Kip1 levels, whereas dominant negative F-box deleted mutant (ΔF-Skp-2) Skp-2 increased p27Kip1 levels. Furthermore, hyperphosphorylation of retinoblastoma protein and SMC proliferation were also reciprocally affected by wild-type and dominant negative Skp-2. Skp-2 expression was absolutely dependent on cell attachment to the ECM and was inhibited by laminin and type-1 fibrillar collagen but increased by fibronectin. Expression of Skp-2 protein, but not mRNA, was associated with focal adhesion kinase (FAK) activity and inhibited by overexpression of FAK-related non-kinase and a dominant negative FAKY397F mutant. Furthermore, the inhibition of Skp-2 expression by dominant negative FAK was reversed by the proteasome inhibitor MG-132. Taken together, these data demonstrate that the vascular ECM controls SMC proliferation via FAK-dependent regulation of Skp-2 protein stability.

Tissue inhibitor of metalloproteinases-3 (TIMP3) is one of four members of a family of proteins that were originally classified according to their ability to inhibit matrix metalloproteinases (MMP). TIMP3, which encodes a potent angiogenesis inhibitor, is mutated in Sorsby fundus dystrophy, a macular degenerative disease with submacular choroidal neovascularization. In this study we demonstrate the ability of TIMP3 to inhibit vascular endothelial factor (VEGF)-mediated angiogenesis and identify the potential mechanism by which this occurs: TIMP3 blocks the binding of VEGF to VEGF receptor-2 and inhibits downstream signaling and angiogenesis. This property seems to be independent of its MMP-inhibitory activity, indicating a new function for this molecule.

Metalloproteinase secretion by macrophages is believed to play a key role in the matrix degradation that underlies atherosclerotic plaque instability and aneurysm formation. We studied the hypothesis that nuclear factor-κB (NF-κB), a transcription factor, is necessary for metalloproteinase secretion and, hence, is a target for pharmacological intervention. Adenovirus-mediated gene transfer of the inhibitory NF-κB subunit, I-κ Bα, was achieved into human monocyte-derived macrophages in vitro and into foam cells produced in vivo in cholesterol-fed rabbits. Human macrophages and rabbit foam cells secreted matrix-degrading metalloproteinase (MMP)-9 without further stimulation, and this was not inhibited by I-κBα (11±16% and 8±10%, respectively;P> 0.05). MMP-1 secretion from human macrophages increased in response to recombinant human CD40 ligand and was inhibited 92±5% by I-κBα (n=3,PPP

The tissue inhibitors of metalloproteinases (TIMPs) are a family of four secreted inhibitors of matrix metalloproteinases (MMPs). Recently, additional functions have been attributed to the TIMPs, including cell growth and inhibition of angiogenesis. In particular, we demonstrated that TIMP-3 overexpression using gene transfer induces apoptosis in a variety of cell types and can inhibit vascular neointima formation in vivo. However, little is know about the mechanisms underlying TIMP-3-mediated apoptosis. Here, using both purified recombinant proteins and novel adenoviral vectors we demonstrate that the prodeath domain of TIMP-3 is located within the N-terminal three loops of TIMP-3. Although both wild type and N-terminal TIMP-3 proteins promoted apoptosis, a T-2/T-3 chimera, in which the N-terminal three loops of TIMP-3 are replaced by those of TIMP-2, failed to induce cell death. Furthermore, a point mutation at residue 1 of TIMP-3 totally abolished MMP-inhibitory activity of TIMP-3 and also failed to promote apoptosis. This study demonstrates, using multiple apoptosis assays, that the prodeath function of TIMP-3 is located within the N-terminal three loops and the presence of functional metalloproteinase-inhibitory activity is associated with the induction of apoptosis.

Lower English literacy achievement of deaf students is often hypothesized to be an impediment for successful adult life experiences. Yet, literacy practices that individuals engage in throughout their daily lives are much more complex than what school-based measures of English can capture and particularly so for deaf individuals. A national large-scale data set with a sample of over 1,000 deaf youths was used to assess what, precisely, standardized measures of literacy may predict in terms of postschool outcomes in three domains: life, employment, and education. Regression analyses indicate that these measures predicted some postschool outcomes, but not all, and if significant, only a small amount of variation in the outcomes was explained. Findings suggest that English literacy, particularly the narrow conceptualization of English literacy skills that are measured through school-based assessments, may not play a significant role in the lives of deaf individuals, contrary to expectations.

Students who are deaf or hard of hearing (SDHH) are a low-incidence group of students; however, SDHH also have a high incidence of additional disabilities (SDHH+). Many SDHH and SDHH+ require accommodations for equal access to classroom instruction and assessment, particularly in mainstreamed educational settings where spoken English is the primary language. Accommodations for SDHH, overall, have increased under federal legislation including the Individuals with Disabilities Education Improvement Act and the No Child Left Behind Act. Unfortunately, specific practice recommendations for SDHH+ and their unique needs are often lacking in the research literature. This article presents findings regarding accommodations use by SDHH and SDHH+ from the National Longitudinal Transition Study 2. Initial logistic regression analysis found no differences in accommodations use of SDHH and SDHH+. However, logistic regression analysis that compared specific additional disability groups with the larger overall SDHH group did find differences in accommodations use for two SDHH+ groups: students who had a learning disability and students with attention deficit hyperactivity disorder. This article includes a discussion of the implications of these findings for both research and practice.

Cell adhesion is essential for cell cycle progression in most normal cells. Loss of adhesion dependence is a hallmark of cellular transformation. The F-box protein Skp2 (S-phase kinase-associated protein 2) controls G1–S-phase progression and is subject to adhesion-dependent transcriptional regulation, although the mechanisms are poorly understood. We identify two cross-species conserved binding elements for the STAF (selenocysteine tRNA gene transcription-activating factor) in the Skp2 promoter that are essential for Skp2 promoter activity. Endogenous STAF specifically binds these elements in EMSA (electrophoretic mobility-shift assay) and ChIP (chromatin immunoprecipitation) analysis. STAF is sufficient and necessary for Skp2 promoter activity since exogenous STAF activates promoter activity and expression and STAF siRNA (small interfering RNA) inhibits Skp2 promoter activity, mRNA and protein expression and cell proliferation. Furthermore, ectopic Skp2 expression completely reverses the inhibitory effects of STAF silencing on proliferation. Importantly, STAF expression and binding to the Skp2 promoter is adhesion-dependent and associated with adhesion-dependent Skp2 expression in non-transformed cells. Ectopic STAF rescues Skp2 expression in suspension cells. Taken together, these results demonstrate that STAF is essential and sufficient for Skp2 promoter activity and plays a role in the adhesion-dependent expression of Skp2 and ultimately cell proliferation.

Background The over-production of superoxide (O 2 − ) derived from NADPH oxidase (NOX) plays a central role in cardiovascular diseases. By contrast, nitric oxide (NO) and prostacyclin (PGI 2 ) are vasculoprotective. The effect of the NO donor, NONOate and iloprost on O 2 − formation, p47 phox and Rac 1 activation in human vascular smooth muscle cells (hVSMCs) was investigated. Methods hVSMCs were incubated with 10nM thromboxane A 2 analogue, U46619 for 16h, and then with apocynin (a NOX inhibitor), NONOate or iloprost for 1h and O 2 − measured spectrophometrically. The role of cyclic AMP and cyclic GMP was examined by co-incubation of drugs with protein kinase (PK) A and G inhibitors listed above. Rac 1 was studied using pull-down assays. Results NONOate and iloprost inhibited O 2 − formation, acutely, effects blocked by inhibition of PKG and PKA, respectively. Rac 1 and p47 phox activation and translocation to the plasma membrane was completely inhibited by NONOate and iloprost, effects again reversed by co-incubation with PKG or PKA inhibitors. Conclusions NO and PGI 2 block the acute activity of NOX in hVSMCs via the cGMP–PKG axis (for NO) and by the cAMP–PKA axis (for iloprost) through inhibition of Rac 1 and p47 phox translocation. These findings have implications in the pathophysiology and treatment of CVD.

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 p27 Kip1 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.

Smooth muscle cell (SMC) proliferation is suppressed in intact blood vessels but stimulated in atherosclerosis, restenosis after angioplasty, and vein graft disease. The cyclin-dependent kinase inhibitors, including p27(Kip1), play important roles in maintaining SMC quiescence. Levels of p27(Kip1) are dependent on attachment to and the composition of the extracellular matrix (ECM). Here we sought to elucidate mechanisms underlying the ECM-dependent regulation of p27(Kip1) and hence, SMC proliferation. Serum stimulation decreased p27(Kip1) levels in isolated SMC but not in rat aorta. The effect was post-translational and mediated by proteasomal degradation. We studied the S-phase-associated kinase protein-2 (Skp-2), an F-box protein involved in ubiquitination and proteasome-mediated degradation. Skp-2 protein is strongly induced by serum from undetectable levels in isolated SMCs but remains undetectable in aorta; Skp-2 mRNA is also lower in aorta. Overexpression of wild-type Skp-2 in SMCs decreased p27(Kip1) levels, whereas dominant negative F-box deleted mutant (DeltaF-Skp-2) Skp-2 increased p27(Kip1) levels. Furthermore, hyperphosphorylation of retinoblastoma protein and SMC proliferation were also reciprocally affected by wild-type and dominant negative Skp-2. Skp-2 expression was absolutely dependent on cell attachment to the ECM and was inhibited by laminin and type-1 fibrillar collagen but increased by fibronectin. Expression of Skp-2 protein, but not mRNA, was associated with focal adhesion kinase (FAK) activity and inhibited by overexpression of FAK-related non-kinase and a dominant negative FAK(Y397F) mutant. Furthermore, the inhibition of Skp-2 expression by dominant negative FAK was reversed by the proteasome inhibitor MG-132. Taken together, these data demonstrate that the vascular ECM controls SMC proliferation via FAK-dependent regulation of Skp-2 protein stability.