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Purpose The Lumi-Solve photo-angioplasty drug eluting balloon catheter (DEBc) may afford safety advantages over current DEBc. Lumi-Solve utilises the guidewire (GW) port and lumen to deliver fibre-optic UV365nm light to the angioplasty balloon which may be problematic. We explore and evaluate alternative Lumi-Solve design options to circumvent fibre-optic use of the GW port and lumen which may enhance efficacy and clinical utility. Methods Effects of guidewire shadowing (GWS) on visible and UV365nm light transmission were evaluated and modelled in-silico. To evaluate the effect of a dedicated intra-balloon fibre-optic port, modified angioplasty balloons and sections of translucent polyethylene terephthalate (PET) GW port tubing were utilised. Investigation of the effect of GWS on chemical and biological photo-activation of balloon surface drug was performed utilising LCMS analysis and inhibition of histone deacetylase activity (HDACi) was measured in human umbilical vein endothelial cells (HUVEC). Results Parallel fibre-optic and GW port configurations generated a GWS of approximately 18.0% of the evaluable balloon surface area and attenuated both visible and UV light intensity by 20.0–25.0% and reduced chemical photo-activation of balloon surface drug and HDACi by at least 40–45%. Alternative fibre-optic port configurations including a spiral design significantly mitigated GWS effects on UV light transmission. Conclusions To avoid use of the GW port and its associated complications a dedicated third port and lumen for the Lumi-Solve fibre-optic may be required. To maximize balloon surface chemical and biological photo-activation, non-parallel, intra-balloon, fibre-optic lumen trajectories, including a spiral design may be useful.

Supplementary Table S1 from Characterisation of the novel apoptotic and therapeutic activities of the histone deacetylase inhibitor romidepsin

Histone deacetylase inhibitors (HDACi) are compounds that target the epigenome and cause tumor cell-selective apoptosis. A large number of these agents that have different chemical structures and can target multiple HDACs are being testing in clinical trials and vorinostat is now an approved drug for the treatment of cutaneous T-cell lymphoma. Although these agents are showing promise for the treatment of hematologic malignancies, it is possible that different drugs may have different mechanistic, biological, and therapeutic activities. When comparing an HDACi belonging to the hydroxamic acid class of compounds (vorinostat) with a cyclic tetrapeptide (romidepsin), we showed that these agents regulate the expression of a common set of cellular genes, but certain genes specifically responded to each agent. Using the Eμ-myc mouse model of B-cell lymphoma, we showed previously that overexpression of the prosurvival proteins Bcl-2 and Bcl-XL inhibited the apoptotic and therapeutic activities of the vorinostat. Herein, we compared and contrasted the apoptotic-inducing activities of the hydroxamic acid oxamflatin with romidepsin. Like vorinostat, oxamflatin was unable to kill lymphomas overexpressing Bcl-2 and Bcl-XL, indicating that these proteins can generally protect cells against this class of HDACi. In contrast, romidepsin was able to induce apoptosis in lymphomas overexpressing Bcl-2 with delayed kinetics of cell death and could mediate therapeutic responses against these lymphomas. However, romidepsin was inactive when Bcl-XL was overexpressed. These data provide strong support that HDACi of different chemical classes may have subtle yet potentially important differences in their molecular and biological activities. [Mol Cancer Ther 2008;7(5):1066–79]

Paclitaxel (PTX)-coated drug eluting balloon catheters (DEBc) used in the management of neointimal hyperplasia (NIH) have been associated with safety concerns. Alternative coating agents and targeted delivery systems may improve safety and DEBc efficacy. Utilizing a multi-platform approach we designed, developed and evaluated Lumi-Solve, a novel DEBc, coated with ultraviolet (UV) 365 nm-activated caged metacept-3 (c-MCT-3), an epigenetic agent from the histone deacetylase inhibitor (HDACi) class. In vitro catheter and contrast media transmission of UV365nm was evaluated spectroscopically. UV365nm conversion of c-MCT-3 to MCT-3 was evaluated chromatographically. Cellular toxicity and HDACi activity of c-MCT-3 ∓UV365nm was evaluated in vitro. In vivo UV365nm conversion of c-MCT-3 to MCT-3 was evaluated in an ovine carotid artery model. Catheter material and dilute contrast media did not attenuate UV365nm transmission or c-MCT-3 activation. c-MCT-3 demonstrated less cellular toxicity than MCT-3 and PTX. UV365nm-activated c-MCT-3 demonstrated HDACi activity. In vivo activation of c-MCT-3 produced MCT-3. Lumi-Solve, a novel DEBc device developed utilizing a combination of chemical, fibre-optic and catheter based technology platforms, demonstrated potential for targeted delivery of bioactive HDACi to the blood vessel wall supporting direct application to the management of NIH and warranting additional in vivo studies.

Drug-eluting balloon catheters (DEBc) coated with paclitaxel (PTX) have been associated with potential safety concerns. An efficacious but less toxic balloon coating may reduce these outcomes. We evaluated a novel DEBc, Epi-Solve, coated with metacept-3 (MCT-3), a member of the histone deacetylase inhibitor (HDACi) class of epigenetic agents, in a large animal model of neointimal hyperplasia (NIH). Plain balloon angioplasty (PABA) catheters were ultrasonically coated with MCT-3 to generate Epi-Solve DEBc. An ovine model of NIH formation was established utilising partial left common carotid artery (LCA) ligation. Twenty-eight days post neointima (NI) induction, PABA, Epi-Solve or PTX-coated DEBc were deployed at the site of induced NI formation. Twenty-eight days post-intervention, ligated vessels were evaluated for attenuation of NI formation, gene expression profiles and immunohistochemical analysis. Epi-Solve DEBc demonstrated attenuation of NIH over no intervention and a trend to inhibition of NIH over PABA. Gene expression analysis and immunohistochemical studies identified significant anti-proliferative and anti-inflammatory signatures and reduced vascular endothelial cell activation compared to PABA. Epi-Solve is a novel HDACi-coated DEBc which demonstrates significant anti-proliferative and anti-inflammatory signatures and reduced vascular endothelial cell activation compared to PABA in an ovine model and may afford endothelial protection.

Objective To explore the potential relationship between the presence of abdominal aortic aneurysm and point of care ankle brachial index acquired posterior tibial artery Doppler waveform to inform on a potential novel biomarker of abdominal aortic aneurysm presence. Methods Abdominal aortic aneurysm presence and posterior tibial artery waveform acquired at time of routine point of care ankle brachial index were determined in 182 patients from an abdominal aortic aneurysm evaluation vascular outpatient clinic. Multivariate technical random forest analysis and logistical regression analysis assessed the outcome of abdominal aortic aneurysm presence and included the independent variables of monophasic initial posterior tibial artery waveform and known abdominal aortic aneurysm risk factors. Results Technical random forest analysis produced a model with an accuracy of 0.59. Initial waveform phase was the most important variable included in the model. Logistical regression analysis revealed a statistically significant negative association between initial monophasic posterior tibial artery waveform and abdominal aortic aneurysm presence in patients with ankle brachial index > 0.9. Leave one out cross validation analysis produced a bias-corrected prediction error value of 0.22. Conclusion No robust association between abdominal aortic aneurysm and point of care ankle brachial index acquired posterior tibial artery waveform was found, suggesting that monophasic posterior tibial artery waveform alone may not be a biomarker of abdominal aortic aneurysm presence.

Paclitaxel (PTX)-coated drug eluting balloon catheters (DEBc) used in the management of neointimal hyperplasia (NIH) have been associated with safety concerns. Alternative coating agents and targeted delivery systems may improve safety and DEBc efficacy. Utilizing a multi-platform approach we designed, developed and evaluated Lumi-Solve, a novel DEBc, coated with ultraviolet (UV) 365 nm-activated caged metacept-3 (c-MCT-3), an epigenetic agent from the histone deacetylase inhibitor (HDACi) class.In vitro catheter and contrast media transmission of UV365nm was evaluated spectroscopically. UV365nm conversion of c-MCT-3 to MCT-3 was evaluated chromatographically. Cellular toxicity and HDACi activity of c-MCT-3 ∓UV365nm was evaluated in vitro. In vivo UV365nm conversion of c-MCT-3 to MCT-3 was evaluated in an ovine carotid artery model.Catheter material and dilute contrast media did not attenuate UV365nm transmission or c-MCT-3 activation. c-MCT-3 demonstrated less cellular toxicity than MCT-3 and PTX. UV365nm-activated c-MCT-3 demonstrated HDACi activity. In vivo activation of c-MCT-3 produced MCT-3.Lumi-Solve, a novel DEBc device developed utilizing a combination of chemical, fibre-optic and catheter based technology platforms, demonstrated potential for targeted delivery of bioactive HDACi to the blood vessel wall supporting direct application to the management of NIH and warranting additional in vivo studies.

Summary Background Obesity is a major issue in Australia and globally. Many individuals struggle to maintain weight loss with lifestyle modification, and adjunctive pharmacotherapy may help. Historically, there have been limited pharmacotherapies for managing obesity. In addition, previous treatments such as phentermine-fenfluramine, rimonabant and sibutramine were withdrawn due to safety issues, resulting in lingering safety concerns. Methods This is a narrative review of published data examining four new pharmacotherapy options for weight management in Australia. Of four new therapeutic options, three may be approved in Australia shortly and one — liraglutide 3.0mg — was approved in December 2015. Liraglutide is a glucagon-like peptide-1 receptor agonist that appears to act by increasing satiety and reducing food intake. Lorcaserin is a selective agonist of the serotonin 2C receptor, which mediates anorectic activity. The naltrexone/bupropion extended release (ER) combination utilises synergistic effects of the two component drugs, mediated via neurons in the hypothalamus, to reduce energy intake. Phentermine/topiramate ER combines the appetite suppressant phentermine with topiramate, an anti-epileptic with appetite-suppressant effects. All can result in meaningful improvements in obesity-related diseases, including diabetes and cardiovascular disorders) in large phase 3 trials, with efficacy demonstrated over 3 years for liraglutide 3.0 mg and 1–2 years for the rest. Conclusions The landscape of obesity treatment is changing rapidly. Of the new therapeutic options presented, all options have associated adverse events requiring long-term safety data, but the availability of new options is a welcome development.

Background It has been suggested that diabetes medications, such as metformin, may have effects that inhibit abdominal aortic aneurysm (AAA) growth. The aim of this study was to examine the association of diabetes treatments with AAA growth in three patient cohorts. Methods AAA growth was studied using ultrasound surveillance in cohort 1, repeated CT in cohort 2 and more detailed repeat CT in cohort 3. Growth was estimated by the mean annual increase in maximum AAA diameter. Results A total of 1697 patients with an AAA were studied, of whom 118, 39 and 16 patients were prescribed metformin for the treatment of diabetes in cohorts 1, 2 and 3 respectively. Prescription of metformin was associated with a reduced likelihood of median or greater AAA growth in all three cohorts (cohort 1: adjusted odds ratio (OR) 0·59, 95 per cent c.i. 0·39 to 0·87, P = 0·008; cohort 2: adjusted OR 0·38, 0·18 to 0·80, P = 0·011; cohort 3: adjusted OR 0·13, 0·03 to 0·61, P = 0·010). No other diabetes treatment was significantly associated with AAA growth in any cohort. Conclusion These findings suggest a potential role for metformin in limiting AAA growth.

Recent evidence suggests an association exists between resistance to epigenetic therapy (EGT) and the expression of programmed cell death protein 1 (PD-1) and programmed death-ligand 1 (PD-L1) in myeloid malignancies. Biomarkers are required to predict resistance to EGT in myeloid malignancies, which together with the delineation of associated molecular mechanisms, may provide additional understanding for novel treatment strategies when investigating resistance to EGT. The present study aimed to investigate the in vivo effects of EGT on the expression of PD-1, PD-L1 and orphan nuclear receptor NUR77 with clinical responses in patients with myeloid malignancies. In addition, in vitro and in vivo characterization of the effects of EGT on the expression of NF-κB and Bcl-xL, potential downstream targets of PD-L1 reverse signaling, were evaluated to determine components of the molecular mechanism responsible for these effects. The in vivo effects of EGT on the expression of PD-1, PD-L1 and a previously identified molecular marker of response to EGT, NUR77 was characterized in peripheral blood mononuclear cells (PBMC) from patients with myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) treated with either azacytidine (Aza) alone or a combination of Aza and the histone deacetylase inhibitor (HDACi) LBH-589 during the first cycle of therapy. The correlation of clinical responses to treatment with EGT with the expression of PD-1, PD-L1 and NUR77 demonstrated that the induction of PD-L1 mRNA levels was associated with resistance to EGT despite the concurrent augmentation of NUR77 expression. The characterization of potential downstream effector molecules of reverse PD-L1 signaling identified EGT-mediated induction of Bcl-xL and NF-κB mRNA expression in vitro and in vivo, suggesting a potential anti-apoptotic molecular mechanism was responsible for PD-L1-mediated resistance to EGT. Taken together, these observations suggest that enhanced PD-L1 expression may confer resistance to EGT over known EGT response markers in myeloid malignancies, and provides a potential molecular mechanism involving the modulation of effectors of PD-L1 reverse signaling, which may in-part, be responsible for these effects.

Hydroxamic acid-based histone deacetylase inhibitors (HDACi) are a class of epigenetic agents with potentially broad therapeutic application to several disease states including post angioplasty mediated neointimal hyperplasia (NIH). Precise spatiotemporal control over the release of HDACi at the target blood vessel site is required for the safe and successful therapeutic use of HDACi in the setting of drug eluting balloon catheter (DEBc) angioplasty treatment of NIH. We aimed to develop and characterise a novel photoactive HDACi, as a potential coating agent for DEBc. Metacept-3 1 was caged with a photo-labile protecting group (PPG) to synthesise a novel UV365nm active HDACi, caged metacept-3 15. Conversion of caged metacept-3 15 to active/native metacept-3 1 by UV365nm was achievable in significant quantities and at UV365nm power levels in the milliwatt (mW) range. In vitro evaluation of the biological activity of pre and post UV365nm activation of caged metacept-3 15 identified significant HDACi activity in samples exposed to short duration, mW range UV365nm. Toxicity studies performed in human umbilical vein endothelial cells (HUVEC's) identified significantly reduced toxicity of caged metacept-3 15 pre UV365nm exposure compared with native metacept-3 1 and paclitaxel (PTX). Taken together these findings identify a novel photo-activated HDACi, caged metacept-3 15, with pharmacokinetic activation characteristics and biological properties which may make it suitable for evaluation as a novel coating for targeted DEBc angioplasty interventions.

Sodium glucose transporter type 2 inhibitors may reduce cardiovascular events in type 2 diabetes. Our study aimed to determine the effect of the sodium glucose transporter type 2 inhibitor dapagliflozin on endothelial cell activation, vasoreactivity and atherogenesis using in vitro and in vivo models and identify associated molecular mechanisms.In vitro studies utilised human vascular endothelial cells stimulated with tumour necrosis factor α or hyperglycaemic conditions. In vivo studies were performed in C57Bl/6J mice to evaluate direct vasorelaxation responses evoked by acute dapagliflozin administration and acute vaso-protective effects of dapagliflozin on hyperglycaemia-induced endothelial dysfunction. Adult and aged Apolipoprotein E-deficient mice maintained on a high-fat diet were used to investigate endothelial-dependent vascular reactivity and atherogenesis. Dapagliflozin treatment (1.0 mg/kg/day) was administered for 4 weeks.In vitro studies demonstrated dapagliflozin-mediated attenuation of tumour necrosis factor α- and hyperglycaemia-induced increases in intercellular adhesion molecule-1, vascular cell adhesion molecule-1, plasminogen activator inhibitor type 1 and NFκB expression. Acute dapagliflozin administration dose-dependently induced endothelium-independent vasorelaxation. Chronic dapagliflozin treatment improved endothelial function and significantly reduced in vivo vascular adhesion molecule and phospho-IκB expression together with macrophage vessel wall infiltration.These observations identify a potential role for dapagliflozin in the attenuation of atherogenesis and identify anti-inflammatory molecular mechanisms associated with these effects.

Neointimal hyperplasia (NIH), a pathophysiological event identified in bypass graft and stent re-stenosis, is characterised by aberrant vascular smooth muscle cell (VSMC) migration and proliferation. Recent evidence identifies histone deacetylase modulation as a regulator of VSMC proliferation and migration and a potential therapeutic target in the treatment of NIH. The purpose of our study was to determine the in vitro and in vivo potential of a novel agent, MCT-3, to modulate VSMC migration, proliferation and NIH.In vitro VSMC studies utilized reverse transcriptase and real time Q-PCR gene expression analysis, western blot, elisa assay and cellular proliferation and migration scratch assay's. In vivo studies utilized the partial carotid artery ligation model of NIH together with immunohistochemistry in FVB/N mice.MCT-3 treatment induced histone H3 and H4 acetylation and inhibited VSMC migration and proliferation in vitro and significantly attenuated NIH in vivo. MCT-3-mediated regulation of orphan nuclear receptor NUR77, Plasminogen Activator Inhibitor Type-1 (PAI-1) and cyclin dependent kinase inhibitors (CDKI) p21(CIP1/WAF1) and p27(KIP1) expression was also identified.Together these observations identify a novel agent, MCT-3, with histone deacetylase inhibitory activity, able to inhibit NIH and identify a potential molecular mechanism responsible for these effects. Additional pre-clinical studies may be warranted to determine the potential clinical utility of this compound.

Epigenetic regulation of microRNA (miRNA) expression has recently been implicated in the pathogenesis of myelodysplastic syndrome (MDS). Particular interest has focused on miRNA-124 expression, which is inhibited in MDS and has recently been demonstrated to be upregulated in response to epigenetic treatment (EGT). Previous studies have determined the in vitro and in vivo expression of miRNA-124 and several molecular targets, including cyclin-dependent kinase (CDK) 4, CDK6 and enhancer of zeste homolog 2 (EZH2), in order to elucidate the molecular mechanisms associated with the miRNA-124-mediated therapeutic response to EGT in MDS and identify additional potential biomarkers of early EGT treatment response in myeloid malignancies. In vitro studies in the HL60 leukemic cell line identified upregulation of miRNA-124 expression in response to single-agent EGT with either azacytidine (AZA) or the histone deacetylase inhibitor panobinostat (LBH589). Combination EGT with AZA and LBH589 resulted in significant additive induction of miRNA-124 expression. Expression of downstream targets of miRNA-124, including CDK4, CDK6 and EZH2, in response to single agent and combined EGT was determined in HL60 cells. Single and combination EGT treatment resulted in inhibition of CDK4, CDK6 and EZH2 expression with combination EGT resulting in a significant and additive inhibitory effect. In vivo studies using peripheral blood mononuclear cells from patients receiving combination EGT for high risk MDS or acute myeloid leukemia demonstrated significant induction of miRNA-124 and inhibition CDK4 and CDK6 messenger (m)RNA expression in patients that responded to combination EGT. A trend to inhibited EZH2 mRNA expression was also identified in response to combination EGT. Overall, the present observations identify a potential molecular mechanism for miRNA-124-mediated response to EGT involving regulation of CDK4, CDK6 and EZH2 expression. In addition, the present findings further qualify miRNA-124 as a possible biomarker of early response to EGT in myeloid malignancies and potentially a valid therapeutic target, together with CDK4, CDK6 and EZH2.

A couple of recent studies suggest that DNA methyltransferase inhibitors, currently used in the treatment of hematologic cancers, increase the sensitivity of tumor cells to immune-checkpoint–inhibitor therapy.

Experimental studies suggest that angiotensin II plays a central role in the pathogenesis of abdominal aortic aneurysm. This trial aims to evaluate the efficacy of the angiotensin receptor blocker telmisartan in limiting the progression of abdominal aortic aneurysm.Telmisartan in the management of abdominal aortic aneurysm (TEDY) is a multicentre, parallel-design, randomised, double-blind, placebo-controlled trial with an intention-to-treat analysis. We aim to randomly assign 300 participants with small abdominal aortic aneurysm to either 40 mg of telmisartan or identical placebo and follow patients over 2 years. The primary endpoint will be abdominal aortic aneurysm growth as measured by 1) maximum infra-renal aortic volume on computed tomographic angiography, 2) maximum orthogonal diameter on computed tomographic angiography, and 3) maximum diameter on ultrasound. Secondary endpoints include change in resting brachial blood pressure, abdominal aortic aneurysm biomarker profile and health-related quality of life. TEDY is an international collaboration conducted from major vascular centres in Australia, the United States and the Netherlands.Currently, no medication has been convincingly demonstrated to limit abdominal aortic aneurysm progression. TEDY will examine the potential of a promising treatment strategy for patients with small abdominal aortic aneurysms.Australian and Leiden study centres: Australian New Zealand Clinical Trials Registry ACTRN12611000931976 , registered on 30 August 2011; Stanford study centre: clinicaltrials.gov NCT01683084 , registered on 5 September 2012.

The glucagon like peptide-1 receptor (GLP-1R) agonist liraglutide attenuates induction of plasminogen activator inhibitor type-1 (PAI-1) and vascular adhesion molecule (VAM) expression in human vascular endothelial cells (hVECs) in vitro and may afford protection against endothelial cell dysfunction (ECD), an early abnormality in diabetic vascular disease. Our study aimed to establish the dependence of the in vitro effects of liraglutide on the GLP-1R and characterise its in vivo effects in a mouse model of ECD. In vitro studies utilised the human vascular endothelial cell line C11-STH and enzyme-linked immunosorbent assays (ELISA) for determination of PAI-1 and VAM expression. In vivo studies of vascular reactivity and immunohistochemical analysis were performed in the ApoE-/- mouse model. In vitro studies demonstrated GLP-1R-dependent liraglutide-mediated inhibition of stimulated PAI-1 and VAM expression. In vivo studies demonstrated significant improvement in endothelial function in liraglutide treated mice, a GLP-1R dependent effect. Liraglutide treatment also increased endothelial nitric oxide synthase (eNOS) and reduced intercellular adhesion molecule-1 (ICAM-1) expression in aortic endothelium, an effect again dependent on the GLP-1R. Together these studies identify in vivo protection, by the GLP-1R agonist liraglutide, against ECD and provide a potential molecular mechanism responsible for these effects.

The thiazolidinediones (TZDs) have been reported to reduce atherogenesis in preclinical models and atherosclerosis in clinical trials in pre-diabetic and diabetic patients. Although peroxisome proliferator-activated receptor γ (PPARγ)-mediated effects on gene expression have been thought responsible for this effect, a complete understanding of the molecular mechanisms responsible remains to be fully elucidated. We have previously reported PPARγ-independent modulation of NUR77 (also known as Nr4a1), an orphan nuclear receptor deemed important in the atherogenic process, in association with TZD-mediated inhibition of tumour necrosis factor α (TNFα) induction of plasminogen activator inhibitor type 1 expression. Here, we report NUR77 mRNA expression is increased in human vascular endothelial cells (HUVEC) stimulated by TNFα and that this effect is inhibited by a TZD in a PPARγ-independent manner. TZD treatment of HUVEC also inhibited the stimulatory effects of TNFα on NUR77 promoter activity, again in a PPARγ-independent manner, confirming the transcriptional nature of this effect. TZD treatment also attenuated the binding of nuclear proteins to the nuclear factor kappa B (NF-κB)-binding site of the NUR77 promoter in HUVEC in a PPARγ-independent manner. In addition, TZD treatment also inhibited TNFα-mediated induction of NF-κB1 mRNA expression. Our results suggest a potential PPARγ-independent molecular mechanism for the anti-atherogenic effects of TZDs involving NF-κB-mediated transcriptional inhibition of cytokine-mediated induction of the orphan nuclear receptor NUR77 in HUVEC.

Abdominal aortic aneurysm (AAA) is a common disease associated with significant cardiovascular morbidity and mortality. The pathogenesis of AAA is poorly defined, making targeting of new therapies problematic. Current evidence favours an interaction of multiple environmental and genetic factors in the initiation and progression of AAA. Epigenetics is the term used to define the properties of the genome that are not explained by the primary sequence, but are due to the modifications of DNA and/or associated proteins. Previous research indicates the association of gene specific promoter DNA hyper-methylation and global DNA hypo-methylation with atherosclerosis. Evidence also suggests an important role for epigenetic processes such as histone acetylation in cardiovascular diseases including atherosclerosis and restenosis. Altered DNA methylation or histone acetylation occur in inflammation, cellular proliferation and remodelling processes and therefore maybe relevant to the pathology of AAA. Important risk factors for AAA, including cigarette smoking, older age, male gender and hypertension, have been linked with epigenetic effects and thus could act in this way to promote AAA. In this review, we discuss the potential role of epigenetic mechanisms in AAA. Since epigenetic alterations are to some extent reversible, further study of this area may identify new treatment targets for AAA.

Earlier we generated novel derivatives of the hydroxamate-based histone deacetylase inhibitor (HDACi), Oxamflatin (Ox), which demonstrate considerable HDACi activity. Here the effects of one such derivative, Metacept-1 (MCT-1), alone or in combination with tamoxifen on mammary tumour growth have been assessed in a syngeneic orthotopic model. MCT-1 alone resulted in a trend towards inhibition of growth of 4T1.2 mammary tumours. Since the combination of MCT-1 and tamoxifen up-regulates estrogen receptor expression in 4T1.2 cells in vitro, we tested this combination and found a significant reduction in primary tumour growth over tamoxifen treatment alone. Taken together, these observations suggest that the novel HDACi MCT-1 may warrant further exploration in the treatment of estrogen receptor positive breast carcinoma, particularly when used in combination with conventional agents such as tamoxifen.

Glucagon-like peptide-1 (GLP-1) administration attenuates endothelial cell dysfunction in diabetic patients and inhibits tumour necrosis factor α (TNF)-mediated plasminogen activator inhibitor type-1 (PAI-1) induction in human vascular endothelial cells. The short half-life of GLP-1 mediated via degradation by the enzyme dipeptidyl peptidase 4 mandates the clinical use of long-acting GLP-1 analogues. The effects of a long-acting GLP-1 analogue on PAI-1 and vascular adhesion molecule expression in vascular endothelial cells are unknown. In this report, we demonstrate for the first time that the treatment with liraglutide, a long-acting GLP-1 analogue, inhibited TNF or hyperglycaemia-mediated induction of PAI-1, intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 mRNA and protein expression in a human vascular endothelial cell line. In addition, treatment attenuated TNF- or hyperglycaemia-mediated induction of the orphan nuclear receptor Nur77 mRNA expression. Taken together, these observations indicate that liraglutide inhibits TNF- or glucose-mediated induction of PAI-1 and vascular adhesion molecule expression, and this effect may involve the modulation of NUR77. These effects suggest that liraglutide may potentially improve the endothelial cell dysfunction associated with premature atherosclerosis identified in type 2 diabetic patients.

Aims Diabetes increases the risk of atherothrombosis, but reduces the risk of abdominal aortic aneurysm (AAA). The reason for this difference is unknown. We examined the role of diabetes and glycation on AAA expansion and extracellular matrix–monocyte interactions. Methods and results We followed 198 patients (20 with diabetes) who had 30–45 mm AAAs with yearly aortic ultrasound for 3 years. Diabetes was independently associated with reduced AAA growth ( β = −0.17, P = 0.01; OR for expansion above median 0.18, 95% confidence interval 0.06–0.57). In vitro incubation of resting human monocytes with glycated bovine serum albumin or monomeric type I collagen increased matrix metalloproteinase (MMP) secretion. In contrast, exposure of activated monocytes to glycated type I collagen lattices induced a marked reduction in MMP and interleukin-6 secretion. This de-activating effect was also demonstrated in cross-linked non-glycated collagen lattices, healthy decellularized aortic media, and decellularized aortic media from diabetes patients with atherosclerosis. In contrast, decellularized aortic media from patients with atherosclerosis, but no diabetes, induced increased MMP secretion. Conclusion These findings confirm that the progression of AAA is slower in patients with diabetes and suggest a mechanism by which the aortic media may be protected from degradation in these individuals.

Glucagon-like peptide-1 (GLP-1) has been proposed as a target for treatment of type 2 diabetes. GLP-1 has also been demonstrated to improve endothelial cell dysfunction in diabetic patients. Elevated plasminogen activator inhibitor type-1 [corrected] (PAI-1) levels have been implicated in endothelial cell dysfunction. The effect of GLP-1 on PAI-1 expression in vascular endothelial cells has not been explored. In a spontaneously transformed human umbilical vein endothelial cell (HUVEC) line, C11-spontaneously transformed HUVEC (STH) and primary HUVEC cells, GLP-1 treatment, in the presence of a dipeptidyl peptidase IV inhibitor, attenuated induction of PAI-1 protein and mRNA expression by tumour necrosis factor-alpha (TNF-alpha). GLP-1 also inhibited the effect of TNF-alpha on a reporter gene construct harbouring the proximal PAI-1 promoter. In addition, GLP-1 attenuated TNF-alpha-mediated induction of Nur77 mRNA and TNF-alpha-mediated binding of nuclear proteins (NPs) to the PAI-1, Nur77, cis-acting response element nerve growth factor induced clone B response element (NBRE). GLP-1 treatment also inhibited TNF-alpha-mediated induction of Akt phosphorylation. Taken together, these observations suggest that GLP-1 inhibits TNF-alpha-mediated PAI-1 induction in vascular endothelial cells, and this effect may involve Akt-mediated signalling events and the modulation of Nur77 expression and NP binding to the PAI-1 NBRE.

Objectives— In vitro and animal studies have implicated osteopontin (OPN) in the pathogenesis of aortic aneurysm. The relationship between serum concentration of OPN and variants of the OPN gene with human abdominal aortic aneurysm (AAA) was investigated. Methods and Results— OPN genotypes were examined in 4227 subjects in which aortic diameter and clinical risk factors were measured. Serum OPN was measured by ELISA in two cohorts of 665 subjects. The concentration of serum OPN was independently associated with the presence of AAA. Odds ratios (and 95% confidence intervals) for upper compared with lower OPN tertiles in predicting presence of AAA were 2.23 (1.29 to 3.85, P =0.004) for the population cohort and 4.08 (1.67 to 10.00, P =0.002) for the referral cohort after adjusting for other risk factors. In 198 patients with complete follow-up of aortic diameter at 3 years, initial serum OPN predicted AAA growth after adjustment for other risk factors (standardized coefficient 0.24, P =0.001). The concentration of OPN in the aortic wall was greater in patients with small AAAs (30 to 50 mm) than those with aortic occlusive disease alone. There was no association between five single nucleotide polymorphisms or haplotypes of the OPN gene and aortic diameter or AAA expansion. Conclusions— Serum and tissue concentrations of OPN are associated with human AAA. We found no relationship between variation of the OPN gene and AAA. OPN may be a useful biomarker for AAA presence and growth.

Background: Glucagon-like peptide-1 receptor agonists may have a role in modulation of cardiac fibrosis. Our study aimed to determine the effect of the glucagon-like peptide-1 receptor agonist liraglutide in obesity, hypertension and age-induced murine models of cardiac fibrosis and identify associated molecular mechanisms. Methods: C57Bl/6J mice on a high-fat diet and C57Bl/6J mice on a normal chow diet treated with angiotensin II were used to induce obesity and hypertension-mediated cardiac fibrosis, respectively. C57Bl/6J mice 20 months old were used to study age-induced cardiac fibrosis. Liraglutide treatment of 30 µg/kg/day–300 µg/kg s.c. twice daily was administered for 4 weeks. Results: Liraglutide treatment attenuated obesity, hypertension and age-induced increases in interstitial cardiac fibrosis and expression of inflammatory and oxidative stress markers. Conclusions: These observations identify a potential role for liraglutide in the prevention of cardiac fibrosis and identify molecular mechanisms associated with these effects.

Glucagon-like peptide-1 (GLP-1) based therapies are used to treat type II diabetes via increasing insulin secretion and inhibiting glucagon production. Recent evidence suggests that activating the GLP-1 receptor may also mediate direct vaso-protective effects. Therefore the objective of the study was to determine whether GLP-1R stimulation conferred cardio- and vaso-protection in a non-diabetic setting using the angiotensin (Ang) II infusion model of hypertension and cardiovascular dysfunction. Male C57Bl/6J mice (4-6 months) were assigned to one of the following 4 week treatment protocols: 1) vehicle (saline), 2) Ang II (800ng/kg/day), 3) Ang II + liraglutide (30μg/kg/day), 4) Ang II + liraglutide (300μg/kg/day). All treatments were administered via osmotic mini-pumps (s.c). After 4 weeks the effect of liraglutide treatment on blood pressure, vascular function and cardiac remodelling was examined. Liraglutide (both doses) attenuated Ang II-induced increase in systolic blood pressure (Ang II: 175.3 ± 8.6mmHg vs Ang II+Lirag (30) 150.2 ± 6.4 mmHg or Ang II+Lirag (300): 145.4 ± 6.9 mmHg) without affecting blood glucose levels. Liraglutide (both doses) completely prevented Ang II-induced endothelial dysfunction (% maximum relaxation: Ang II=50.7 ± 7.8%; Ang II+Lirag (30)=82.7 ± 5.8; Ang II+Lirag (300)=81.5 ± 6.1%). In the heart, liraglutide prevented Ang II-induced cardiomyocyte hypertrophy (n=7-10; p

Background Experimental studies suggest that angiotensin II plays a central role in the pathogenesis of abdominal aortic aneurysm. This trial aims to evaluate the efficacy of the angiotensin receptor blocker telmisartan in limiting the progression of abdominal aortic aneurysm. Methods/Design Telmisartan in the management of abdominal aortic aneurysm (TEDY) is a multicentre, parallel-design, randomised, double-blind, placebo-controlled trial with an intention-to-treat analysis. We aim to randomly assign 300 participants with small abdominal aortic aneurysm to either 40 mg of telmisartan or identical placebo and follow patients over 2 years. The primary endpoint will be abdominal aortic aneurysm growth as measured by 1) maximum infra-renal aortic volume on computed tomographic angiography, 2) maximum orthogonal diameter on computed tomographic angiography, and 3) maximum diameter on ultrasound. Secondary endpoints include change in resting brachial blood pressure, abdominal aortic aneurysm biomarker profile and health-related quality of life. TEDY is an international collaboration conducted from major vascular centres in Australia, the United States and the Netherlands. Discussion Currently, no medication has been convincingly demonstrated to limit abdominal aortic aneurysm progression. TEDY will examine the potential of a promising treatment strategy for patients with small abdominal aortic aneurysms. Trial registration Australian and Leiden study centres: Australian New Zealand Clinical Trials RegistryACTRN12611000931976, registered on 30 August 2011; Stanford study centre: clinicaltrials.govNCT01683084, registered on 5 September 2012.

The plasminogen-activating (PA) and matrix metalloproteinase (MMP) enzyme systems are implicated in proteolytic turnover of the extracellular matrix (ECM) associated with biologic processes including wound healing, inflammation and angiogenesis. Aberrant expression of components of the PA and MMP enzyme systems occurs in the pathogenesis of metastatic cancer. Oxamflatin (Ox), a novel hydroxamic acid derivative, inhibits u-PA mRNA expression and proteolytic activity while simultaneously upregulating the expression of the natural inhibitor of u-PA, plasminogen activator inhibitor type 2 (PAI-2) in metastatic cancer cells. We have characterized the effects of Ox and a novel derivative, Metacept-1 (MCT-1), on PA and MMP-mediated proteolysis and invasion in several metastatic tumor lines. Both compounds are able to inhibit u-PA-, MMP-2- and MMP-9-mediated gene expression at low micromolar concentrations as well as u-PA- and MMP-mediated proteolysis as assessed by zymography, with MCT-1 being the more effective of the 2 agents in some assays. Cellular invasion assays correlate with gene expression and zymography experiments identifying both Ox and MCT-1 as able to inhibit invasion of metastatic cancer cell lines through matrigel at nanomolar concentrations, with MCT-1 more effective than Ox in 2 of the 3 cancer cell lines assessed. © 2004 Wiley-Liss, Inc.

Surgeons are increasingly being exposed to the term 'hyperhomocysteinaemia' but few understand this condition that affects up to 10% of the population, or its pathological sequelae. Hyperhomocysteinaemia has been identified as an important and independent risk factor for atherosclerosis. There is increasing evidence that, in addition to coronary disease, hyperhomocysteinaemia is also associated with an increased risk of developing peripheral arterial disease. Causes of elevated homocysteine levels include inherited enzyme deficiencies and acquired vitamin deficiencies. Detection of hyperhomocysteinaemia is particularly relevant in patients with early onset atherosclerosis. Effective lowering of elevated homocysteine levels is possible with folate, vitamin B6 and B12 supplementation and may reduce the incidence and sequelae of atherosclerotic peripheral arterial disease.

Combination epigenetic treatment (EGT) utilizing DNA methyl transferase inhibitors (DNMTi) and histone deacetylase inhibitors (HDACi) may be more efficacious than single agent treatment in myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). The molecular mechanisms behind the potential clinical efficacy of combination EGT treatment are incompletely understood and the frequently lengthy EGT regimes required to determine clinical response have generated a significant demand for early molecular markers of treatment response. Our study aimed to identify the effect of combination azacitidine (AZA) and panobinostat (LBH589) on expression levels of a panel of genes implicated in the pathogenesis of high-risk MDS or AML in HL-60 cells. We also characterized gene expression profiles in peripheral blood mononuclear (PBMCs) from patients in a recently reported phase Ib/II clinical trial using the combination of AZA and LBH589 and correlated these findings with clinical response to treatment. In vitro analysis demonstrated increased expression of caspase-3, Nor-1, NUR77, p15INK4B and p21WAF1/CIP1 and decreased expression of Bcl‑xL in HL-60 cells treated with combination EGT. Analysis of patient samples prior to treatment demonstrated a significant reduction in NUR77 and p21WAF1/CIP1 expression compared to healthy controls. NUR77 and p21WAF1/CIP1 levels were similar between treatment non‑responders and responders at screening. Early post first cycle treatment (day 25) analysis demonstrated a significant increase in expression of both NUR77, and p21WAF1/CIP1. A significant increase in NUR77, and p21WAF1/CIP1 together with a trend to increase in p15INK4B first cycle expression was observed in treatment responders compared to non-responders. In summary, combination AZA and LBH589 epigenetic treatment is associated with in vitro and in vivo modulation of genes implicated in the pathogenesis of MDS/AML. Early expression of NUR77 and p21WAF1/CIP1 correlated with clinical response to combination EGT suggesting investigation for potential use as molecular markers of early treatment response may be warranted.

Objectives: To compare the potency, toxicity and mechanism of action of multiple histone deacetylase inhibitors (HDACi) in activating HIV production from latency. Design: In-vitro analysis of HDACi in a primary T-cell model of HIV latency and latently infected cell lines. Methods: Latently infected chemokine ligand 19 (CCL19)-treated CD4 T cells and the latently infected cell lines ACH2 and J-Lat were treated with a panel of HDACi, including entinostat, vorinostat, panonbinostat and MCT3. Viral production and cell viability were compared. Expression of cellular HDACs was measured by western blot and PCR. Association of HDACs with the HIV long-terminal repeat (LTR) using latently infected CCL19-treated primary CD4 T cells in the presence and absence of specific HDACi was determined by chromatin immunoprecipitation (ChIP). Results: We demonstrated considerable variation in the potency and toxicity of HDACi in latently infected primary CD4 T cells and cell lines. All HDACi tested activated HIV production in latently infected primary T cells with greatest potency demonstrated with entinostat and vorinostat and greatest toxicity with panobinostat. Following the addition of HDACi in vitro, there were no changes in markers of T-cell activation or expression of the HIV coreceptors chemokine (C-X-C motif) receptor 4 (CXCR4) or chemokine (C-C motif) receptor type 5 (CCR5). ChIP analysis of latently infected CCL19-treated primary CD4 T cells showed binding by HDAC1, HDAC2 and HDAC3 to the LTR with removal of HDAC1 and HDAC2 following treatment with the HDACi vorinostat and HDAC1 only following treatment with entinostat. Conclusion: The HDACi entinostat, selective for inhibition of class I HDACs, induced virus expression in latently infected primary CD4 T cells making this compound an attractive novel option for future clinical trials.

Presented in part at the 53rd Annual Meeting of the American Society of Hematology, San Diego, CA, December 10-13, 2011, and the 17th Congress of the European Haematology Association, Amsterdam, June 14-17, 2012. Therapeutic options are limited for elderly patients with acute myeloid leukemia (AML). A phase Ib/II study was undertaken to evaluate the maximum-tolerated dose (MTD) and preliminary efficacy of the pan-histone deacetylase inhibitor panobinostat (LBH589) in combination with azacitidine in patients with AML or high-risk myelodysplastic syndrome (MDS) naïve to intensive chemotherapy. Thirty-nine patients (AML=29, MDS=10) received azacitidine 75 mg/m2 subcutaneously (days 1-5) and oral panobinostat (starting on day 5, thrice weekly for seven doses) in 28-day cycles until toxicity or disease progression. Dose-limiting toxicities during the phase Ib stage were observed in 0/4 patients receiving 10 mg panobinostat, in 1/7 patients (fatigue) receiving 20 mg, in 1/6 patients (fatigue) receiving 30 mg and in 4/5 patients (fatigue, syncope, hyponatremia and somnolence) receiving 40 mg. In phase II, an additional 17 patients received panobinostat at a MTD of 30 mg. The overall response rate (ORR=CR+CRi+PR) in patients with AML was 31% (9/29) and that in patients with MDS was 50% (5/10). After a median follow-up of 13 months, the median overall survival was 8 and 16 months in patients with AML and MDS, respectively. Increased histone H3 and H4 acetylation was a useful early biomarker of clinical response. Combining panobinostat with azacitidine was tolerable and clinically active in high-risk MDS/AML patients, warranting further exploration. Refereed/Peer-reviewed

Plasminogen-activator inhibitor type 2 (PAI-2), a serine protease inhibitor involved in the regulation of urokinase-dependent proteolysis, is also implicated in the inhibition of tumor-necrosis-factor-(TNF)-mediated apoptosis. The PAI-2 gene is one of the most TNF-responsive genes known and is also highly induced by the phorbol ester phorbol 12-myristate 13-acetate (PMA) and the phosphatase inhibitor, okadaic acid, in both HT-1080 fibrosarcoma and U-937 histiocytic cells. We sought to identify and characterize regulatory cis-acting DNA elements and trans -acting factors which mediate basal and inducible PAI-2 gene transcription. A series of promoter deletion mutants (nucleotides –1859 to –91) fused to the chloramphenicol acetyl transferase (CAT) reporter gene were transfected into HT-1080 cells. Two repressor regions were identified; one distally between positions –1859 and –1100, and one proximally between positions –259 and –219. Cells transfected with constructs harboring more than 259 bp promoter sequence produced a 10–15-fold increase in CAT activity when treated with PMA or okadaic acid, but produced only a minimal (2.5-fold) increase in response to TNF. Removal of the proximal repressor by deletion to position –219, or by internal deletion from the –1100 PAI-2 CAT construct, resulted in a selective increase in TNF responsiveness, suggesting that induction of PAI-2 gene transcription by TNF is associated with derepression. Detailed analysis of the proximal repressor utilizing the electrophoretic mobility shift assay (EMSA), identified two novel and distinct protein-binding sites (A and B). Site A is located within the 40-bp proximal repressor while site B is situated immediately adjacent to the 3′ boundary. Treatment of cells with PMA or okadaic acid produced no change in the binding activity of proteins recognising sites A or B. However, treatment of cells with TNF results in a profound selective reduction in site-B-binding activity, suggesting that this site plays a significant role in TNF-mediated regulation of PAI-2 gene expression. Our findings suggest that TNF-mediated induction of PAI-2 gene expression involves derepression and is associated with cis -acting and trans -acting factors located within and adjacent to the proximal repressor region.

Summary PAI-2, the most enigmatic member of the serine protease inhibitor gene superfamily, is generally recognised as an inhibitor of urokinase (u-PA) and, to a lesser extent, tissue-type plasminogen activator. The role of plasminogen activator inhibitor type-2 (PAI-2) in the regulation of a u-PA-mediated proteolysis is well established. However, the predominantly cytosolic location of PAI-2 has raised much speculation as to the role this serpin plays in intracellular proteolysis and in particular in the events leading to cellular differentiation and apoptosis. In vitro studies have highlighted the fact that PAI-2 is one of the most tumour necrosis factor (TNF) responsive genes so far described suggesting that PAI-2 may also play a significant role in TNF biology.

The potential atheroprotective effects of glucagon-like peptide-1 (GLP-1), long-acting GLP-1 analogues and inhibitors of the enzyme dipeptidyl peptidase 4 (DPP-4) are currently the subject of intense research. Recent evidence suggests the effects of DPP-IV inhibitors, may, in-part, be mediated by GLP-1 independent molecular mechanisms. In this report we demonstrate that treatment of human vascular endothelial cells with the DPP-IV inhibitor sitagliptin inhibited tumour necrosis factor alpha (TNFα) induction of plasminogen activator inhibitor type-1 (PAI-1), intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) mRNA and protein expression and that this effect was observed to be both GLP-1-dependent and independent. Importantly we identify a molecular mechanism involving sitagliptin-mediated attenuation of TNFα-mediated induction of NFκB and orphan nuclear receptor NUR77 mRNA expression, also able to be reproduced, in part, independent of GLP-1. Taken together these observations may serve to provide a molecular explanation, involving transcriptional regulation of gene expression, for recent in vivo studies suggesting DPP-IV inhibitors may have novel, GLP-1 independent, effects in acting to attenuate endothelial cell dysfunction and atherogenesis.

Histone deacetylase inhibitors (HDACi) demonstrate considerable in vitro and in vivo activity and clinical efficacy in the treatment of hematological malignancies. Pre-clinical and early phase clinical trials identify therapeutic activity using a combination of HDACi and demethylating agents which may be more efficacious than single agent treatment. Our studies aimed to determine the effects and molecular mechanisms of action of novel hydroxamate (MCT-3) and benzamide [MGCD0103 (MG)] HDACi's in the HL-60 cell line alone and in combination with the demethylating agent 5-aza-cytidine (AZA). MG, MCT-3 and AZA treatment significantly inhibited HL-60 cell growth in vitro with MG being the most potent agent. MG in combination with AZA demonstrated no significant increase in inhibition of cell growth over MG treatment alone whilst MCT-3 in combination with AZA demonstrated increased inhibition of cell growth over either agent alone although no more significant than MG alone. MG alone or MCT-3 in combination with AZA significantly increased p15 and caspase-3 expression. MG and MCT-3 significantly attenuated AZA-induced MMP-9 mRNA expression and proteolytic activity. Interestingly, MCT-3, MG and AZA alone and in combination increased expression of the novel tumour suppressor gene Nur77, important in leukemogenesis, with MG a more potent inducer as a single agent. These observations suggest the enhanced anti-leukemia activity of the combination of AZA and HDACi may only reside with certain HDACi classes and may be in-part explained by regulation of genes associated with cell cycle arrest, apoptosis and tumour suppression.

Glucagon-like peptide-1 (GLP-1) has been proposed as a target for treatment of type 2 diabetes. GLP-1 has also been demonstrated to improve endothelial cell dysfunction in diabetic patients. Elevated plasminogen activator inhibitor type-1 [corrected] (PAI-1) levels have been implicated in endothelial cell dysfunction. The effect of GLP-1 on PAI-1 expression in vascular endothelial cells has not been explored. In a spontaneously transformed human umbilical vein endothelial cell (HUVEC) line, C11-spontaneously transformed HUVEC (STH) and primary HUVEC cells, GLP-1 treatment, in the presence of a dipeptidyl peptidase IV inhibitor, attenuated induction of PAI-1 protein and mRNA expression by tumour necrosis factor-alpha (TNF-alpha). GLP-1 also inhibited the effect of TNF-alpha on a reporter gene construct harbouring the proximal PAI-1 promoter. In addition, GLP-1 attenuated TNF-alpha-mediated induction of Nur77 mRNA and TNF-alpha-mediated binding of nuclear proteins (NPs) to the PAI-1, Nur77, cis-acting response element nerve growth factor induced clone B response element (NBRE). GLP-1 treatment also inhibited TNF-alpha-mediated induction of Akt phosphorylation. Taken together, these observations suggest that GLP-1 inhibits TNF-alpha-mediated PAI-1 induction in vascular endothelial cells, and this effect may involve Akt-mediated signalling events and the modulation of Nur77 expression and NP binding to the PAI-1 NBRE.

Conformational analogues of the hydroxamic acid Oxamflatin 1—compounds 3a, 3b and 4—have been synthesised to enable evaluation of the impact of varying the linking section on histone deacetylase inhibition. Preliminary testing indicates treatment of leukaemia cells with each of the analogues leads to significant inhibition of histone deacetylase and reduction in cell growth and proliferation.

Increased plasminogen activator inhibitor type 1 (PAI-1) levels are observed in endothelial cells stimulated by tumour necrosis factor alpha (TNFalpha). Thiazolidinediones (TZDs) may inhibit elevated endothelial cell PAI-1 accounting, in part, for the putative atheroprotective effects of TZDs. In an endothelial cell line, Rosiglitazone (RG) and Pioglitazone (PG) inhibited induction of PAI-1 by TNFalpha. The specific peroxisome proliferator-activated receptor gamma (PPARgamma) inhibitor, SR-202, failed to modulate this effect. RG also inhibited the effect of TNFalpha on a reporter gene construct harbouring the proximal PAI-1 promoter and PAI-1 mRNA in cells co-transfected with a dominant-negative PPARgamma construct. RG and PG attenuated TNFalpha-mediated induction of trans-acting factor(s) Nur77/Nurr1 and binding of nuclear proteins (NP) to the cis-acting element (NBRE). SR-202 failed to modulate these effects. The observations suggest TZDs inhibit TNFalpha-mediated PAI-1 induction independent of inducible PPARgamma activation and this may involve in the modulation of Nur77/Nurr1 expression and NP binding to the PAI-1 NBRE.

Histone deacetylase inhibitors (HDACIs) are a new class of chemotherapeutic drugs able to induce tumor cell apoptosis and/or cell cycle arrest; however, the molecular mechanisms underpinning their anticancer effects are poorly understood. Herein, we assessed the apoptotic pathways activated by three HDACIs, suberoylanilide hydroxamic acid, oxamflatin, and depsipeptide. We determined that all three drugs induced the accumulation of cells with a 4n DNA content and apoptosis mediated by the intrinsic apoptotic pathway. HDACI-induced mitochondrial membrane damage and apoptosis were inhibited by overexpression of Bcl-2, but not by the polycaspase inhibitor N-tert-butoxy-carbonyl-Val-Ala-Asp-fluoromethylketone (zVAD-fmk). Moreover, induction of a G(1)-S checkpoint through overexpression of p16(INK4A) or suppression of de novo protein synthesis also inhibited HDACI-induced cell death. Proteolytic cleavage of caspase-2, which is poorly inhibited by zVAD-fmk, was concomitant with HDACI-induced death; however, full processing of caspase-2 to the p19 active form was blocked by Bcl-2. Whereas all three drugs induce the activation of the proapoptotic Bcl-2 protein Bid upstream of mitochondrial membrane disruption, Bid cleavage in response to depsipeptide was significantly attenuated by zVAD-fmk. Suberoylanilide hydroxamic acid and oxamflatin could kill both P-glycoprotein (P-gp)(+) MDR cells and their P-gp(-) counterparts, whereas depsipeptide was shown to be a substrate for P-gp and was less effective in killing P-gp(+) cells. These data provide insight into the functional profile of three HDACIs and are important for the development of more rational approaches to chemotherapy, where information regarding the genetic profile of the tumor is matched with the functional profile of a given chemotherapeutic drug to promote favorable clinical responses.

Surgeons are increasingly being exposed to the term 'hyperhomocysteinaemia' but few understand this condition that affects up to 10% of the population, or its pathological sequelae. Hyperhomocysteinaemia has been identified as an important and independent risk factor for atherosclerosis. There is increasing evidence that, in addition to coronary disease, hyperhomocysteinaemia is also associated with an increased risk of developing peripheral arterial disease. Causes of elevated homocysteine levels include inherited enzyme deficiencies and acquired vitamin deficiencies. Detection of hyperhomocysteinaemia is particularly relevant in patients with early onset atherosclerosis. Effective lowering of elevated homocysteine levels is possible with folate, vitamin B6 and B12 supplementation and may reduce the incidence and sequelae of atherosclerotic peripheral arterial disease.

Clopidogrel is a widely used anti-platelet agent for the prevention of arterial thrombosis. Clopidogrel is administered as a pro-drug and metabolised to its active metabolite by the hepatic cytochrome P450 2C19 (CYP2C19) enzyme. The active metabolite is responsible for the anti-platelet activity of clopidogrel. Recent studies demonstrate that single nucleotide polymorphisms, (SNP's), in the gene for CYP2C19 result in significantly reduced production of the active metabolite of clopidogrel. Additional studies demonstrate that patients with SNP's in the CYP2C19 gene, including CYP2C19*2,*3,*4, and *5, have reduced production of the active metabolite of clopidogrel, reduced inhibition of platelet aggregation and increased incidence of coronary, cerebrovascular, and coronary stent thrombosis. We have been interested in determining the CYP2C19 genotype in cases of coronary stent thrombosis whilst on clopidogrel treatment and provide two case reports of coronary stent thrombosis whilst taking clopidogrel with subsequent CYP2C19 genotyping. As patients at risk of atherothrombosis in general, and stent thrombosis in particular, may be receiving or considered for anti-platelet therapy including clopidogrel, genotyping for CYP2C19 SNP's may be of benefit in the selection of appropriate anti-platelet therapy.

Cell surface, urokinase (u-PA)-mediated, plasminogen activation has recently been recognised as a process integral to extracellular matrix degradation. The primary inhibitor of u-PA activity in the extracellular matrix is plasminogen activator inhibitor type 2 (PAI-2), a serine protease inhibitor. The malignant metastatic phenotype is associated with excessive and uncontrolled, tumour cell-associated, u-PA-mediated, extracellular matrix degradation. Inhibition of the malignant metastatic phenotype via induction of PAI-2 expression and/or inhibition of u-PA expression may represent a novel means via which the metastatic phenotype can be arrested. Agents capable of inducing PAI-2 and/or inhibiting u-PA activity may restrict u-PA-mediated tumour cell proteolysis and facilitate in the development of therapeutic strategies to combat malignant disease. We have identified the hydroxamic acid derivative oxamflatin, previously noted to revert the malignant phenotype in K-ras-transformed NIH-3T3 cells, as capable of upregulating PAI-2 and simultaneously suppressing u-PA expression in two different cell systems. In addition, zymographic analysis indicated that oxamflatin treatment results in a significant reduction in u-PA proteolytic activity in both HT-1080 fibrosarcoma and U-937 histiocytic lymphoma cells. We postulate that oxamflatin represents a novel means by which induction of PAI-2 and concomitant inhibition of u-PA gene and protein expression can be achieved and may be of benefit in inhibiting the malignant metastatic phenotype.

Since its discovery over a decade ago, evidence has accumulated implicating the cell-surface urokinase receptor (u-PAR), in numerous biological processes. Most notable has been the identification of a critical role for u-PAR in the regulation of cell-surface plasminogen activation in physiological and pathological conditions. Recent evidence suggests that u-PAR, a glycosylphosphatidylinositol-linked receptor, lacking transmembrane and cytoplasmic domains, is also involved in processes not related to plasminogen activation, including cellular adhesion and the transmission of extracellular signals across the plasma membrane. Involvement of activated u-PAR in these events identifies previously unsuspected roles for this molecule and defines a new field of research in u-PAR biology. We discuss the molecular biology of u-PAR together with the underlying mechanisms responsible for the novel functional roles recently ascribed to this pleiotropic molecule.