Connective tissue growth factor/CCN2 stimulates actin disassembly through Akt/protein kinase B-mediated phosphorylation and cytoplasmic translocation of p27(Kip-1)

Connective tissue growth factor (CTGF/CCN2) is a 38-kDa secreted protein, a prototypic member of the CCN family, which is up-regulated in many diseases, including atherosclerosis, pulmonary fibrosis, and diabetic nephropathy. We previously showed that CTGF can cause actin disassembly with concurrent down-regulation of the small GTPase Rho A and proposed an integrated signaling network connecting focal adhesion dissolution and actin disassembly with cell polarization and migration. Here, we further delineate the role of CTGF in cell migration and actin disassembly in human mesangial cells, a primary target in the development of renal glomerulosclerosis. The functional response of mesangial cells to treatment with CTGF was associated with the phosphorylation of Akt/protein kinase B (PKB) and resultant phosphorylation of a number of Akt/PKB substrates. Two of these substrates were identified as FKHR and p27(Kip-1). CTGF stimulated the phosphorylation and cytoplasmic translocation of p27(Kip-1) on serine 10. Addition of the PI-3 kinase inhibitor LY294002 abrogated this response; moreover, addition of the Akt/PKB inhibitor interleukin (IL)-6-hydroxymethyl-chiro-inositol-2(R)-2-methyl-3-O-octadecylcarbonate prevented p27(Kip-1) phosphorylation in response to CTGF. Immunocytochemistry revealed that serine 10 phosphorylated p27(Kip-1) colocalized with the ends of actin filaments in cells treated with CTGF. Further investigation of other Akt/PKB sites on p27(Kip-1), revealed that phosphorylation on threonine 157 was necessary for CTGF mediated p27(Kip-1) cytoplasmic localization; mutation of the threonine 157 site prevented cytoplasmic localization, protected against actin disassembly and inhibited cell migration. CTGF also stimulated an increased association between Rho A and p27(Kip-1). Interestingly, this resulted in an increase in phosphorylation of LIM kinase and subsequent phosphorylation of cofilin, suggesting that CTGF mediated p27(Kip-1) activation results in uncoupling of the Rho A/LIM kinase/cofilin pathway. Confirming the central role of Akt/PKB, CTGF-stimulated actin depolymerization only in wild-type mouse embryonic fibroblasts (MEFs) compared to Akt-1/3 (PKB alpha/gamma) knockout MEFs. These data reveal important mechanistic insights into how CTGF may contribute to mesangial cell dysfunction in the diabetic milieu and sheds new light on the proposed role of p27(Kip-1) as a mediator of actin rearrangement.

Plasma glutathione peroxidase activity is reduced in haemodialysis patients

Cardiovascular disease is the major cause of morbidity and mortality in patients with end-stage renal failure. Increased free radical production and antioxidant depletion may contribute to the greatly increased risk of atherosclerosis in these patients. Glutathione peroxidase (GPX) is an important antioxidant, the plasma form of which is synthesized mainly in the kidney (eGPX). The aim of this study was to assess the activity of eGPX in patients with end-stage renal failure on haemodialysis. Venous blood was collected from 87 haemodialysis patients immediately prior to and after dialysis and from 70 healthy controls. Serum eGPX activity was measured using hydrogen peroxide as substrate and immunoreactivity determined by ELISA. eGPX activity was significantly reduced in dialysis patients when compared to controls (106 +/- 2.7 and 281 +/- 3.6 U/l respectively, p <0.001). Following haemodialysis, eGPX activity rose significantly to 146 +/- 3.8 U/l, p <0.001, although remaining below control values (p <0.005). Immunoreactive eGPX, however, was similar in all groups (pre-dialysis 14.10 +/- 1.26 microg/ml, post-dialysis 14.58 +/- 1.35 microg/ml, controls 15.20 +/- 1.62 microg/ml, p = NS). A decrease was observed in the specific activity of eGPX in patients when compared to controls (8.81 +/- 1.14, 10.71 +/- 1.54 and 21.97 +/- 1.68 U/mg respectively, p <0.0001). eGPX activity is impaired in patients undergoing haemodialysis and so may contribute to atherogenesis in renal failure.

Arsenic levels in the soils and macrophytes of the 'Entremuros' after the Aznalcóllar mine spill

In April 1998, a holding lagoon containing pyrite ore processing waste rich in arsenic, failed and released 5-6 million m(3) of highly polluting sludge and acidic water. Over 2700 ha of the internationally important Doñana National and Natural Parks were contaminated. The area of Natural Park to sustain the greatest impact was known as the Entremuros. This paper presents 0-5 cm soil monitoring data from the Entremuros, from sampling campaigns 6 and 18 months after the disaster; as well as macrophyte root, rhizome and stem data from samples taken 18 months after the spill. Results show a clear, decreasing, north-south arsenic soil pollution trend, both 6 and 18 months after the spill, and suggest a small reduction in total soil arsenic levels occurred over time; although a significant increase in extractable arsenic is also noted. The two macrophytes (Typha dominguensis and Scirpus maritimus) studied herein are not accumulating arsenic in stem parts, however, accumulation of arsenic on iron plaque on the roots of these plants may be occurring. Further work is recommended in order to determine the ecotoxicological significance of this process in relation to the avian food-chains of Doñana, and elsewhere.

Community-living nonagenarians in northern ireland have lower plasma homocysteine but similar methylenetetrahydrofolate reductase thermolabile genotype prevalence compared to 70-89-year-old subjects

This cross-sectional study assessed relationships between plasma homocysteine, 'thermolabile' methylenetetrahydrofolatereductase (MTHFR) genotype, B vitamin status and measures of renal function in elderly (70-89 years) and nonagenarian (90+ years) subjects, with the hypothesis that octo/nonagenarian subjects who remain healthy into old age as defined by 'Senieur' status might show reduced genetic or environmental risk factors usually associated with hyperhomocysteinaemia. Plasma homocysteine was 9.1 micromol/l (geometric mean [GM]) for all elderly subjects. Intriguingly, homocysteine was significantly lower in 90+ (GM; 8.2 micromol/l) compared to 70-89-year-old subjects (GM; 9.8 micromol/l) despite significantly lower glomerular filtration rate (GFR) and serum B12 in nonagenarian subjects and comparable MTHFR thermolabile (TT) genotype frequency, folate and B6 status to 70-89-year-olds. For all elderly subjects, the odds ratio and 95% confidence intervals for plasma homocysteine being in the highest versus lowest quartile was 4.27 (2.04-8.92) for age 90 years, 3.4 (1.5-7.8) for serum folate 10.7nmol/l, 3.0 (0.9-10.2) for creatinine >140 compared

Glutathione peroxidase 1 genotype is associated with an increased risk of coronary artery disease

Objective Oxidative stress is implicated in the pathogenesis of many human diseases including atherosclerosis. Human glutathione peroxidase 1 (hgpx1) participates in limiting cellular damage caused by oxidation. A characteristic polyalanine sequence polymorphism in exon 1 of hgpx1 produces three alleles with five, six or seven alanine (ALA) repeats in this sequence. The objective of this study was to determine whether hgpx1 genotype is associated with an altered risk of coronary artery disease (CAD).

Methods The frequency of the ALA6 allele was determined in 207 men with angiographic evidence of significant CAD compared to a control group (n = 146), by analysing the lengths of polymerase chain reaction fragments containing the ALA repeat polymorphism. Additional information was collected on severity of CAD, presence or absence of a prior acute myocardial infarction (AMI), smoking status, body mass index (BMI) and other clinical data.

Results There was a significant association between individuals with at least one ALA6 allele and an increased risk of CAD after adjustment for age, BMI and smoking status (odds ratio, 2.07, 95% confidence interval, 1.08-3.99, P = 0.029). However, there was no association between hgpx1 genotype and a previous history of AMI or hgpx1 genotype and severity of CAD.

Conclusion We conclude that individuals possessing one or two ALA6 alleles appear to be at a modest increased risk of CAD. This observation merits further investigation in other patient populations.

XBP1 mRNA splicing triggers an autophagic response in endothelial cells through BECLIN-1 transcriptional activation

Sustained activation of X-box-binding protein 1 (XBP1) results in endothelial cell (EC) apoptosis and atherosclerosis development. The present study provides evidence that XBP1 mRNA splicing triggered an autophagic response in ECs by inducing autophagic vesicle formation and markers of autophagy BECLIN-1 and microtubule-associated protein 1 light chain 3ß (LC3-ßII). Endostatin activated autophagic gene expression through XBP1 mRNA splicing in an inositol-requiring enzyme 1a (IRE1a)-dependent manner. Knockdown of XBP1 or IRE1a by shRNA in ECs ablated endostatin-induced autophagosome formation. Importantly, data from arterial vessels from XBP1 EC conditional knock-out (XBP1eko) mice demonstrated that XBP1 deficiency in ECs reduced the basal level of LC3ß expression and ablated response to endostatin. Chromatin immunoprecipitation assays further revealed that the spliced XBP1 isoform bound directly to the BECLIN-1 promoter at the region from nt -537 to -755. BECLIN-1 deficiency in ECs abolished the XBP1-induced autophagy response, whereas spliced XBP1 did not induce transcriptional activation of a truncated BECLIN-1 promoter. These results suggest that XBP1 mRNA splicing triggers an autophagic signal pathway through transcriptional regulation of BECLIN-1.

Epigenetics in Vascular Disease - Therapeutic Potential of New Agents

Vascular diseases, including atherosclerosis, angioplasty-induced restenosis, vessel graft arteriosclerosis and hypertension-related stenosis, remain the most prevalent cause of death in the developed world. The aetiology of vascular diseases is multifactorial with both genetic and environmental factors. Recently, some of the most promising research identifies the epigenetic modification of the genome to play a major role in the disease development, linking the environmental insults with gene regulation. In this process, modification of DNA by methylation, and histone modification by acetylation, methylation, phosphorylation and/or SUMOylation are reported. Importantly, recent studies demonstrated that histone deacetylase (HDAC) enzymes are crucial in endothelial integrity, smooth muscle proliferation and in the formation of arteriosclerosis in animal models. The study of HDACs has shown remarkable specificity of HDAC family members in vascular cell growth/death that influences the disease process. Interestingly, the effects of HDACs on arteriosclerosis development in animal models have been observed after HDAC inhibition using specific inhibitors. This provides a new approach for the treatment of vascular disease using the agents that influence the epigenetic process in vascular cells. This review updates the rapid advances in epigenetics of vascular diseases focusing on the role of HDAC family in atherosclerosis. It will also discuss the underlying mechanisms of histone acetylation in vascular cells and highlight the therapeutic potential of such agents.

Role of histone deacetylases in vascular cell homeostasis and arteriosclerosis

Histone deacetylases (HDACs) are a family of enzymes that remove acetyl groups from lysine residues of histone proteins, a modification that results in epigenetic modulation of gene expression. Although originally shown to be involved in cancer and neurological disease, HDACs are also found to play crucial roles in arteriosclerosis. This review summarizes the effects of HDACs and HDAC inhibitors on proliferation, migration, and apoptosis of endothelial and smooth muscle cells. In addition, an updated discussion of HDACs' recently discovered effects on stem cell differentiation and atherosclerosis is provided. Overall, HDACs appear to be promising therapeutic targets for the treatment of arteriosclerosis and other cardiovascular diseases.

Cavitary melanoma of the ciliary body. A study of eight cases

Purpose: The authors present the unique clinical features of cavitary uveal melanoma. Design: Retrospective chart review. Participants: Eight patients with cavitary uveal melanoma. Main Outcome Measures: The clinical, ultrasonographic, and histopathologic features of eight patients with cavitary melanoma of the ciliary body were studied. Results: In all eyes there was a brown ciliary body mass that blocked transmission of light on trans-scleral transillumination. Ocular ultrasonography revealed a large, single hollow cavity (unilocular 'pseudocyst') in five cases and multiple hollow cavities (multilocular 'pseudocyst') in three cases. The cavity occupied a mean of 55% of the entire mass thickness (range, 31%-79%). In five cases, a basal uveal mass was noted on ultrasonography. Four patients underwent tumor resection; one had enucleation, and three had 1251 radioactive plaque treatment. In the five cases confirmed histopathologically, the cavitation was empty, contained erythrocytes, serous fluid, and/or pigment-laden macrophages. In no case was the cavity lined by necrotic tumor, endothelial cells, or epithelial cells. Conclusion: Ciliary body melanoma can develop an intralesional cavity resembling an intraocular cyst. The presence of a solid mass at the base and a thick wall surrounding the cavity can assist in the differentiation of cavitary melanoma from benign cyst.

Transpupillary thermotherapy for choroidal melanoma:Tumor control and visual results in 100 consecutive cases

Objective: The authors evaluated the results of primary transpupillary thermotherapy for choroidal melanoma in 100 cases. Design: Prospective nonrandomized analysis of treatment method. Participants: One hundred patients with choroidal melanoma were studied. Main Outcome Measures: Tumor response, ocular side effects, and visual results. Results: Of 100 consecutive patients with choroidal melanoma treated with transpupillary thermotherapy, the mean tumor basal diameter was 7.1 mm and tumor thickness was 2.8 mm. The tumor margin touched the optic disc in 34 eyes (34%) and was beneath the fovea in 42 eyes (42%). Documented growth was present in 64 eyes (64%), and known clinical risks for growth were present in all of the remaining 36 eyes (36%), with an average of 4 of 5 statistical risk factors for growth per tumor. After a mean of three treatment sessions and 14 months of follow-up, the mean tumor thickness was reduced to 1.4 mm. Treatment was successful in 94 eyes (94%) and failed in 6 eyes (6%). Three patients with amelanotic tumors showed no initial response to thermotherapy, but subsequent intravenous indocyanine green administration during thermotherapy resulted in improved heat absorption and tumor regression to a flat scar. The six eyes classified as treatment failures included four eyes with tumors that showed partial or no response to thermotherapy, thus requiring plaque radiotherapy or enucleation, and two eyes with recurrence, subsequently controlled with additional thermotherapy. After treatment, the visual acuity was the same (within 1 line) or better than the pretreatment visual acuity in 58 eyes (58%) and worse in 42 eyes (42%). The main reasons for poorer vision included treatment through the foveola for subfoveal tumor (25 eyes), retinal traction (10 eyes), retinal vascular obstruction (5 eyes), optic disc edema (1 eye), and unrelated ocular ischemia (1 eye). Temporal location (versus nasal and superior, P = 0.02) and greater distance from the optic disc (P = 0.04) were risks for retinal traction. Conclusions: Transpupillary thermotherapy may be an effective treatment for small posterior choroidal melanoma, especially those near the optic disc and fovea. Despite satisfactory local tumor control, ocular side effects can result in decreased vision. Longer follow- up will be necessary to assess the impact of thermotherapy on ultimate local tumor control and metastatic disease.

Microneedle-mediated transdermal bacteriophage delivery

Interest in bacteriophages as therapeutic agents has recently been reawakened. Parenteral delivery is the most routinely-employed method of administration. However, injection of phages has numerous disadvantages, such as the requirement of a health professional for administration and the possibility of cross-contamination. Transdermal delivery offers one potential means of overcoming many of these problems. The present study utilized a novel poly (carbonate) (PC) hollow microneedle (MN) device for the transdermal delivery of Escherichia coli-specific 14 bacteriophages both in vitro and in vivo. MN successfully achieved bacteriophage delivery in vitro across dermatomed and full thickness skin. A concentration of 2.67 x 10(6) PFU/ml (plaque forming units per ml) was detected in the receiver compartment when delivered across dermatomed skin and 4.0 x 10(3) PFU/ml was detected in the receiver compartment when delivered across full thickness skin. An in vivo study resulted in 4.13 x 10(3) PFU/ml being detected in blood 30 min following initial MN-mediated phage administration. Clearance occurred rapidly, with phages being completely cleared from the systemic circulation within 24 h, which was expected in the absence of infection. We have shown here that MN-mediated delivery allows successful systemic phage absorption. Accordingly, bacteriophage-based therapeutics may now have an alternative route for systemic delivery. Once fully-investigated, this could lead to more widespread investigation of these interesting therapeutic viruses. (c) 2012 Elsevier B.V. All rights reserved.

Genetic screening protocol for familial hypercholesterolemia which includes splicing defects gives an improved mutation detection rate

Familial hypercholesterolemia (FH) is a common single gene disorder, which predisposes to coronary artery disease. In a previous study, we have shown that in patients with definite FH around 20% had no identifiable gene defect after screening the entire exon coding area of the low density lipoprotein receptor (LDLR) and testing for the common Apolipoprotein B (ApoB) R3500Q mutation. In this study, we have extended the screen to additional families and have included the non-coding intron splice regions of the gene. In families with definite FH (tendon xanthoma present, n = 68) the improved genetic screening protocol increased the detection rate of mutations to 87%. This high detection rate greatly enhances the potential value of this test as part of a clinical screening program for FH. In contrast, the use of a limited screen in patients with possible FH (n = 130) resulted in a detection rate of 26%, but this is still of significant benefit in diagnosis of this genetic condition. We have also shown that 14% of LDLR defects are due to splice site mutations and that the most frequent splice mutation in our series (c.1845 + 11 c > g) is expressed at the RNA level. In addition, DNA samples from the patients in whom no LDLR or ApoB gene mutations were found, were sequenced for the NARC-1 gene. No mutations were identified which suggests that the role of NARC-1 in causing FH is minor. In a small proportion of families (