Dual Role of CREB in The Regulation of VSMC Proliferation: Mode of Activation Determines Pro- or Anti-Mitogenic Function.

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.

Systematic identification of genetic influences on methylation across the human life course.

BACKGROUND: The influence of genetic variation on complex diseases is potentially mediated through a range of highly dynamic epigenetic processes exhibiting temporal variation during development and later life. Here we present a catalogue of the genetic influences on DNA methylation (methylation quantitative trait loci (mQTL)) at five different life stages in human blood: children at birth, childhood, adolescence and their mothers during pregnancy and middle age. RESULTS: We show that genetic effects on methylation are highly stable across the life course and that developmental change in the genetic contribution to variation in methylation occurs primarily through increases in environmental or stochastic effects. Though we map a large proportion of the cis-acting genetic variation, a much larger component of genetic effects influencing methylation are acting in trans. However, only 7 % of discovered mQTL are trans-effects, suggesting that the trans component is highly polygenic. Finally, we estimate the contribution of mQTL to variation in complex traits and infer that methylation may have a causal role consistent with an infinitesimal model in which many methylation sites each have a small influence, amounting to a large overall contribution. CONCLUSIONS: DNA methylation contains a significant heritable component that remains consistent across the lifespan. Our results suggest that the genetic component of methylation may have a causal role in complex traits. The database of mQTL presented here provide a rich resource for those interested in investigating the role of methylation in disease.

The Hippo pathway mediates inhibition of vascular smooth muscle cell proliferation by cAMP.

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.

Non coding RNAs in aortic aneurysmal disease.

An aneurysm is a local dilatation of a vessel wall which is >50% its original diameter. Within the spectrum of cardiovascular diseases, aortic aneurysms are among the most challenging to treat. Most patients present acutely after aneurysm rupture or dissection from a previous asymptomatic condition and are managed by open surgical or endovascular repair. In addition, patients may harbor concurrent disease contraindicating surgical intervention. Collectively, these factors have driven the search for alternative methods of identifying, monitoring and treating aortic aneurisms using less invasive approaches. Non-coding RNA (ncRNAs) are emerging as new fundamental regulators of gene expression. The small microRNAs have opened the field of ncRNAs capturing the attention of basic and clinical scientists for their potential to become new therapeutic targets and clinical biomarkers for aortic aneurysm. More recently, long ncRNAs (lncRNAs) have started to be actively investigated, leading to first exciting reports, which further suggest their important and yet largely unexplored contribution to vascular physiology and disease. This review introduces the different ncRNA types and focus at ncRNA roles in aorta aneurysms. We discuss the potential of therapeutic interventions targeting ncRNAs and we describe the research models allowing for mechanistic studies and clinical translation attempts for controlling aneurysm progression. Furthermore, we discuss the potential role of microRNAs and lncRNAs as clinical biomarkers.

cAMP-induced actin cytoskeleton remodelling inhibits MKL1-dependent expression of the chemotactic and pro-proliferative factor, CCN1.

Elevation of intracellular cAMP concentration has numerous vascular protective effects that are in part mediated via actin cytoskeleton-remodelling and subsequent regulation of gene expression. However, the mechanisms are incompletely understood. Here we investigated whether cAMP-induced actin-cytoskeleton remodelling modulates VSMC behaviour by inhibiting expression of CCN1. In cultured rat VSMC, CCN1-silencing significantly inhibited BrdU incorporation and migration in a wound healing assay. Recombinant CCN1 enhanced chemotaxis in a Boyden chamber. Adding db-cAMP, or elevating cAMP using forskolin, significantly inhibited CCN1 mRNA and protein expression in vitro; transcriptional regulation was demonstrated by measuring pre-spliced CCN1 mRNA and CCN1-promoter activity. Forskolin also inhibited CCN1 expression in balloon injured rat carotid arteries in vivo. Inhibiting RhoA activity, which regulates actin-polymerisation, by cAMP-elevation or pharmacologically with C3-transferase, or inhibiting its downstream kinase, ROCK, with Y27632, significantly inhibited CCN1 expression. Conversely, expression of constitutively active RhoA reversed the inhibitory effects of forskolin on CCN1 mRNA. Furthermore, CCN1 mRNA levels were significantly decreased by inhibiting actin-polymerisation with latrunculin B or increased by stimulating actin-polymerisation with Jasplakinolide. We next tested the role of the actin-dependent SRF co-factor, MKL1, in CCN1 expression. Forskolin inhibited nuclear translocation of MKL1 and binding of MKL1 to the CCN1 promoter. Constitutively-active MKL1 enhanced basal promoter activity of wild-type but not SRE-mutated CCN1; and prevented forskolin inhibition. Furthermore, pharmacological MKL-inhibition with CCG-1423 significantly inhibited CCN1 promoter activity as well as mRNA and protein expression. Our data demonstrates that cAMP-induced actin-cytoskeleton remodelling regulates expression of CCN1 through MKL1: it highlights a novel cAMP-dependent mechanism controlling VSMC behaviour.

Inhibition of Egr1 expression underlies the anti-mitogenic effects of cAMP in vascular smooth muscle cells.

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.

Differences in smoking associated DNA methylation patterns in South Asians and Europeans.

BACKGROUND: DNA methylation is strongly associated with smoking status at multiple sites across the genome. Studies have largely been restricted to European origin individuals yet the greatest increase in smoking is occurring in low income countries, such as the Indian subcontinent. We determined whether there are differences between South Asians and Europeans in smoking related loci, and if a smoking score, combining all smoking related DNA methylation scores, could differentiate smokers from non-smokers. RESULTS: Illumina HM450k BeadChip arrays were performed on 192 samples from the Southall And Brent REvisited (SABRE) cohort. Differential methylation in smokers was identified in 29 individual CpG sites at 18 unique loci. Interaction between smoking status and ethnic group was identified at the AHRR locus. Ethnic differences in DNA methylation were identified in non-smokers at two further loci, 6p21.33 and GNG12. With the exception of GFI1 and MYO1G these differences were largely unaffected by adjustment for cell composition. A smoking score based on methylation profile was constructed. Current smokers were identified with 100% sensitivity and 97% specificity in Europeans and with 80% sensitivity and 95% specificity in South Asians. CONCLUSIONS: Differences in ethnic groups were identified in both single CpG sites and combined smoking score. The smoking score is a valuable tool for identification of true current smoking behaviour. Explanations for ethnic differences in DNA methylation in association with smoking may provide valuable clues to disease pathways.

Migration and DNA methylation: a comparison of methylation patterns in type 2 diabetes susceptibility genes between indians and europeans.

BACKGROUND: Type 2 diabetes is a global problem that is increasingly prevalent in low and middle income countries including India, and is partly attributed to increased urbanisation. Genotype clearly plays a role in type 2 diabetes susceptibility. However, the role of DNA methylation and its interaction with genotype and metabolic measures is poorly understood. This study aimed to establish whether methylation patterns of type 2 diabetes genes differ between distinct Indian and European populations and/or change following rural to urban migration in India. METHODS: Quantitative DNA methylation analysis in Indians and Europeans using Sequenom® EpiTYPER® technology was undertaken in three genes: ADCY5, FTO and KCNJ11. Metabolic measures and genotype data were also analysed. RESULTS: Consistent differences in DNA methylation patterns were observed between Indian and European populations in ADCY5, FTO and KCNJ11. Associations were demonstrated between FTO rs9939609 and BMI and between ADCY5rs17295401 and HDL levels in Europeans. However, these observations were not linked to local variation in DNA methylation levels. No differences in methylation patterns were observed in urban-dwelling migrants compared to their non-migrant rural-dwelling siblings in India. CONCLUSIONS: Analysis of DNA methylation at three type 2 diabetes susceptibility loci highlighted geographical and ethnic differences in methylation patterns. These differences may be attributed to genetic and/or region-specific environmental factors.

Phenotypic and genotypic characterization of coagulase negative staphylococci (CoNS) other than Staphylococcus epidermidis isolated from ocular infections.

PURPOSE: The clinical significance of many species belonging to coagulase-negative staphylococci (CoNS) other than Staphylococcus epidermidis has been increasingly recognized. The present study attempted the characterization of non-S. epidermidis CoNS isolates of ocular origin. METHODS: The isolates were characterized phenotypically by analytical profile index (API) and genotypically by fluorescent amplified fragment length polymorphism (FAFLP). In addition, the presence of genes that are likely to enhance virulence such as biofilm related genes (icaA and icaB) and methicillin resistance (mecA) were detected. RESULTS: API identified seven different species with an identification score varying from 44% to 99%. S. haemolyticus and S. xylosus species identified by API showed good API scores when compared with all other species identified. FAFLP generated 12 clusters from all the isolates and appeared to be more discriminatory. API identification results corresponded well with the FAFLP results only in six clusters. Nearly 40% of isolates showed the presence of mecA and icaAB genes. CONCLUSIONS: Identification results of these two methods corresponded only in 48.4% of the isolates suggesting that the battery of tests using API were not sufficient enough to identify all the species of CoNS. Therefore, it is sensible to rely on two or more identification methods particularly when API species identification has a lower score. FAFLP genotyping appears to be a reliable and rapid alternative identification method for CoNS that can be used in conjunction with any other phenotypic method.

High-resolution genome profiling differentiated Staphylococcus epidermidis isolated from patients with ocular infections and normal individuals.

PURPOSE: To investigate the potential phenotypic and genetic differences among the Staphylococcus epidermidis isolates obtained from control subjects (lower conjunctival sac; n = 14) with those from patients with keratitis (corneal scrapings; n = 18) or endophthalmitis (vitreous; n = 24). METHODS: Biofilm-forming capacity was detected by PCR for the icaAB gene and phenotyping by microtiter plate assay and congo red agar plate. Genotyping was performed by using fluorescence-amplified fragment length polymorphism (FAFLP) and in silico analysis of the FAFLP profiles. RESULTS: Biofilm phenotyping (congo red agar/microtiter plate) differentiated disease-causing strains from control subjects. PCR assays (mecA, icaAB) were not useful in differentiating disease-causing strains from that of control subjects. The biofilm-forming capability appeared more critical in the pathogenesis of keratitis than in that of endophthalmitis. Cluster analysis of FAFLP data generated 11 clusters comprising 4 major clusters (I, II, III, and V) and 7 minor ones. FAFLP analysis clearly showed clustering of most of the commensal isolates in cluster I, separate from keratitis and endophthalmitis isolates. In silico analysis mapped signature bands to genes such as ebh, tagD, ptsI, and sepA, which might have a significant role in transforming less virulent populations of S. epidermidis to more virulent ones. CONCLUSIONS: The population dynamics of S. epidermidis revealed that there are significant genetic variations that can be detected through FAFLP between ocular disease causing isolates and the commensal population.

Activity of newer fluoroquinolones against gram-positive and gram-negative bacteria isolated from ocular infections: an in vitro comparison.

BACKGROUND: To determine the antibacterial activity of newer fluoroquinolones and compare their activity between ciprofloxacin-susceptible and resistant bacterial isolates from patients with keratitis and endophthalmitis. MATERIALS AND METHODS: The minimum inhibitory concentration (MIC) of ciprofloxacin, ofloxacin, levofloxacin, gatifloxacin and moxifloxacin was determined for 123 bacterial isolates, using E test. Among the 123 isolates, 68 were gram-positive (Staphylococcus spp, Streptococcus spp, Corynebacterium spp, Bacillus spp.) and 55 were gram-negative (Pseudomonas aeruginosa). The bacterial isolates were divided into three groups: susceptible/intermediate/resistant to ciprofloxacin. The MIC values for various fluoroquinolones were compared between the three groups and between gram-positive and gram-negative bacteria. RESULTS: For gram-positive isolates, median MICs of fourth generation fluoroquinolones were lower than second generation. The median MIC was lowest for gatifloxacin and moxifloxacin (0.094 mg/ml) in ciprofloxacin-susceptible isolates of gram-positive bacteria. For ciprofloxacin-susceptible gram-negative bacteria, the median MIC of ciprofloxacin (0.19 mg/ml) was significantly lower than ofloxacin, levofloxacin, gatifloxacin and moxifloxacin (1.5, 0.5, 0.5 and 2 mg/ml respectively). Ciprofloxacin-resistant isolates of gram-positive bacteria showed higher MIC of levofloxacin, moxifloxacin and gatifloxacin though they remained susceptible to them. None of the fluoroquinolones were effective against ciprofloxacin-resistant gram-negative bacteria. Overall, for gram-positive bacteria, median MICs of levofloxacin, moxifloxacin and gatifloxacin were below ciprofloxacin, the MIC of gatifloxacin and moxifloxacin was equal for gram-positive bacteria. CONCLUSIONS: Levofloxacin, gatifloxacin and moxifloxacin are statistically more effective against gram-positive bacteria, the latter two being equally effective. Ciprofloxacin remains the most effective fluoroquinolone against gram-negative bacteria.

Fluorescent amplified fragment length polymorphism (FAFLP) genotyping demonstrates the role of biofilm-producing methicillin-resistant periocular Staphylococcus epidermidis strains in postoperative endophthalmitis.

BACKGROUND: An observational case series was used to study the virulence characteristics and genotypes of paired Staphylococcus epidermidis isolates cultured from intraocular samples and from periocular environment of patients with postcataract surgery endophthalmitis. METHODS: Eight S. epidermidis isolates were obtained from three patients (2 from patients #1 and 2 and 4 from patient #3) whose vitreous and/or anterior chamber (AC) specimens and preoperative lid/conjunctiva samples were culture positive. Cultures were identified by API-Staph phenotypic identification system and genotypically characterized by Fluorescent Amplified Fragment Length Polymorphism (FAFLP) and checked for their antimicrobial susceptibility. The isolates were tested for biofilm-production and methicillin-resistance (MR) by PCR amplification of icaAB and mecA gene respectively. RESULTS: Four out of eight S. epidermidis strains showed multiple drug resistance (MDR). All the eight strains were PCR positive for mecA gene whereas seven out of eight strains were positive for icaAB genes. In all three patients FAFLP typing established vitreous isolates of S. epidermidis strains to be indistinguishable from the strains isolated from the patient's conjunctival swabs. However, from patient number three there was one isolate (1030b from lid swab), which appeared to be nonpathogenic and ancestral having minor but significant differences from other three strains from the same patient. This strain also lacked icaAB gene. In silico analysis indicated possible evolution of other strains from this strain in the patient. CONCLUSION: Methicillin-resistant biofilm positive S. epidermidis strains colonizing the conjunctiva and eyelid were responsible for postoperative endophthalmitis (POE).

Diagnosis of herpes simplex virus-1 keratitis: comparison of Giemsa stain, immunofluorescence assay and polymerase chain reaction.

PURPOSE: To evaluate three different diagnostic tests against the gold standard of viral isolation, in the diagnosis of HSV-1 keratitis. METHODS: Corneal scrapings from 170 patients with clinically suspected HSV keratitis were tested by; 1) Giemsa staining procedure for the presence of multinucleated giant cells and lymphocytes, 2) immunofluorescence assay for HSV-1 antigen, 3) polymerase chain reaction (PCR) for HSV-1 DNA and 4) virus isolation by shell vial culture in SIRC (Rabbit corneal epithelial cell line). The results of the former three tests were compared among 14 cases that were culture positive and 156 cases that were culture negative for HSV-1. RESULTS: The sensitivity of PCR was 100%, while IFA and Giemsa had sensitivities of 85.7% and 57.1% respectively. The specificity of PCR, IFA and Giemsa were found to be 67.9%, 85.3% and 85.9% respectively. CONCLUSIONS: In the present study, a combination of PCR and immunofluorescence assay appears to be the most suitable choice of tests for diagnosis of HSV-1 keratitis, while detection of MNGC by Giemsa staining procedure may give us a presumptive diagnosis of suspected viral infection.