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Kappelle P.J.W.H.,University of Groningen | Lambert G.,The Heart Research Institute | Lambert G.,University of Nantes | Dullaart R.P.F.,University of Groningen
Atherosclerosis | Year: 2011

Purpose: Proprotein convertase subtilisin-kexin type 9 (PCSK9) promotes low density lipoprotein (LDL) receptor degradation, thereby providing a key pathway for LDL metabolism. PCSK9 mRNA expression may be upregulated by insulin in murine models. Here we examined effects of exogenous hyperinsulinemia on plasma PCSK9 levels in humans without and with type 2 diabetes mellitus. Methods: A 24. h moderately hyperinsulinemic glucose clamp (30. mU/kg/h) was performed in 8 healthy men and 8 male type 2 diabetic patients. Plasma PCSK9 was measured using a sandwich enzyme-linked immunosorbent assay. Results: Plasma LDL cholesterol and apolipoprotein B were lowered by insulin in healthy subjects and diabetic patients (p< 0.01 for all), whereas triglycerides were also decreased in healthy subjects (p< 0.01). Plasma PCSK9 levels remained unchanged in healthy subjects (median (interquartile range) change, -23 (-63 to 25) %, P = 0.50) and in diabetic patients (change, 4 (-17 to 44) %, P = 0.20). Individual absolute and relative changes in LDL cholesterol, apolipoprotein B and triglycerides after 24. h of insulin were unrelated to changes in PCSK9 (P> 0.15 for all). Conclusion: Plasma PCSK9 levels are not increased by exposure to moderate 24. h hyperinsulinemia in healthy and type 2 diabetic individuals. © 2010 Elsevier Ireland Ltd.


Moheimani F.,The Heart Research Institute | Moheimani F.,University of Sydney | Morgan P.E.,The Heart Research Institute | Morgan P.E.,University of Sydney | And 3 more authors.
Biochimica et Biophysica Acta - Molecular Basis of Disease | Year: 2010

People with diabetes experience chronic hyperglycemia and are at a high risk of developing atherosclerosis and microvascular disease. Reactions of glucose, or aldehydes derived from glucose (e.g. methylglyoxal, glyoxal, or glycolaldehyde), with proteins result in glycation that ultimately yield advanced glycation end products (AGE). AGE are present at elevated levels in plasma and atherosclerotic lesions from people with diabetes, and previous in vitro studies have postulated that the presence of these materials is deleterious to cell function. This accumulation of AGE and glycated proteins within cells may arise from either increased formation and/or ineffective removal by cellular proteolytic systems, such as the proteasomes, the major multi-enzyme complex that removes proteins within cells. In this study it is shown that whilst high glucose concentrations fail to modify proteasome enzyme activities in J774A.1 macrophage-like cell extracts, reactive aldehydes enhanced proteasomal enzyme activities. In contrast BSA, pre-treated with high glucose for 8. weeks, inhibited both the chymotrypsin-like and caspase-like activities. BSA glycated using methylglyoxal or glycolaldehyde, also inhibited proteasomal activity though to differing extents. This suppression of proteasome activity by glycated proteins may result in further intracellular accumulation of glycated proteins with subsequent deleterious effects on cellular function. © 2010 Elsevier B.V.


Di Bartolo B.A.,University of New South Wales | Kavurma M.M.,The Heart Research Institute | Kavurma M.M.,University of Sydney
Current Pharmaceutical Design | Year: 2014

Receptor activator of nuclear factor-κB ligand (RANKL) is a member of the tumour necrosis factor family important in bone remodelling. Recent evidence suggest that calcification in the vessel wall is equivalent to mechanisms mediating bone formation. This review highlights the role of RANKL in vascular arterial calcification. Here, the relationship between RANKL, osteoprotegerin (OPG) and tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) is discussed. Furthermore, we focus on the regulatory mechanisms mediating RANKL gene expression and transcription in cells of the vessel wall. A better understanding of RANKL-mediated signalling may help develop more sophisticated cell-based therapies to inhibit calcification of the vessel wall. © 2014 Bentham Science Publishers.


Bhandare A.M.,The Heart Research Institute | Bhandare A.M.,Macquarie University | Kapoor K.,The Heart Research Institute | Kapoor K.,Macquarie University | And 4 more authors.
Journal of Neuroscience | Year: 2016

Cardiovascular autonomic dysfunction in seizure is a major cause of sudden unexpected death in epilepsy. The catecholaminergic neurons in the rostral ventrolateral medulla (RVLM) maintain sympathetic vasomotor tone and blood pressure through their direct excitatory projections to the intermediolateral (IML) cell column. Glutamate, the principal excitatory neurotransmitter in brain, is increased in seizures. Pituitary adenylate cyclase activating polypeptide (PACAP) is an excitatory neuropeptide with neuroprotective properties, whereas microglia are key players in inflammatory responses in CNS. We investigated the roles of glutamate, PACAP, and microglia on RVLM catecholaminergic neurons during the cardiovascular responses to 2 mg/kg kainic acid (KA)-induced seizures in urethane anesthetized, male Sprague Dawley rats. Microinjection of the glutamate antagonist, kynurenic acid (50 nl; 100 mM) into RVLM, blocked the seizure-induced 43.2 ± 12.6% sympathoexcitation (p ≤ 0.05), and abolished the pressor responses, tachycardia, and QT interval prolongation. PACAP or microglia antagonists (50 nl) (PACAP(6–38), 15 pmol; minocycline 10 mg/ml) microinjected bilaterally into RVLM had no effect on seizure-induced sympathoexcitation, pressor responses, or tachycardia but abolished the prolongation of QT interval. The actions of PACAP or microglia on RVLM neurons do not cause sympathoexcitation, but they do elicit proarrhythmogenic changes. An immunohistochemical analysis in 2 and 10 mg/kg KA-induced seizure rats revealed that microglia surrounding catecholaminergic neurons are in a “surveillance” state with no change in the number of M2 microglia (anti-inflammatory). In conclusion, seizure-induced sympathoexcitation is caused by activation of glutamatergic receptors in RVLM that also cause proarrhythmogenic changes mediated by PACAP and microglia. © 2016 the authors.


Jansson P.J.,University of Sydney | Hawkins C.L.,The Heart Research Institute | Lovejoy D.B.,University of Sydney | Richardson D.R.,University of Sydney
Journal of Inorganic Biochemistry | Year: 2010

Iron chelation therapy was initially designed to alleviate the toxic effects of excess iron evident in iron-overload diseases. However, some iron chelator-metal complexes have also gained interest due to their high redox activity and toxicological properties that have potential for cancer chemotherapy. This communication addresses the conflicting results published recently on the ability of the iron chelator, Dp44mT, to induce hydroxyl radical formation upon complexation with iron (B.B. Hasinoff and D. Patel, J Inorg. Biochem. 103 (2009), 1093-1101). This previous study used EPR spin-trapping to show that Dp44mT-iron complexes were not able to generate hydroxyl radicals. Here, we demonstrate the opposite by using the same technique under very similar conditions to show the Dp44mT-iron complex is indeed redox-active and induces hydroxyl radical formation. This was studied directly in an iron(II)/H2O2 reaction system or using a reducing iron(III)/ascorbate system implementing several different buffers at pH 7.4. The demonstration by EPR that the Dp44mT-iron complex is redox-active confirms our previous studies using cyclic voltammetry, ascorbate oxidation, benzoate hydroxylation and a plasmid DNA strand-break assay. We discuss the relevance of the redox activity to the biological effects of Dp44mT. © 2010.


Barter P.,The Heart Research Institute
Atherosclerosis Supplements | Year: 2011

Evidence that low-density lipoprotein-cholesterol (LDL-C) causes cardiovascular disease (CVD) is overwhelming. It has also been proven beyond all doubt that lowering the level of LDL-C using statins reduces CV risk. However, many people remain at high risk even when their level of LDL-C has been reduced by aggressive treatment with statins. One reason for this residual risk can be a low level of high-density lipoprotein-cholesterol (HDL-C).The concentration of HDL-C is an independent, inverse predictor for CVD. This relationship is apparent even when treatment with statins has reduced the level of LDL-C to below 1.8 mmol/L (70 mg/dL). It has therefore been suggested that raising the level of HDL-C should be considered as a therapeutic strategy for reducing the residual CV risk that persists in some people, despite aggressive LDL-C lowering with statins. HDL particles have several functions with the potential to protect against arterial disease, the best known of which relates to their ability to promote cholesterol efflux from macrophages in the artery wall. However, HDLs have several additional protective properties that are independent of their involvement in cholesterol metabolism. For example, they have properties that reduce oxidation, vascular inflammation and thrombosis, improve endothelial function, promote endothelial repair, enhance insulin sensitivity and promote insulin secretion by pancreatic beta islet cells. There is also a large and compelling body of evidence in animal models showing that interventions that increase HDL levels are profoundly anti-atherogenic. Major causes of low HDL are abdominal obesity and type 2 diabetes, the worldwide incidences of which are increasing at alarming rates. Strategies to increase the concentration of HDL should begin with lifestyle changes such as weight reduction, increased physical activity and smoking cessation. However, compliance with such measures is frequently poor and pharmacological intervention may be required. Currently available HDL-raising medications include fibrates, niacin and statins. There is indisputable evidence that lowering LDL-C levels using statins translates into a large reduction in CV risk. There is also mounting evidence that increasing the level of HDL-C using statins contributes to an additional reduction in CV risk. For example, the increase in HDL-C levels that was associated with simvastatin treatment in the 4S study was a significant predictor for the reduction in CV events. Moreover, a meta-analysis of 1,455 patients in 4 coronary intravascular ultrasound imaging trials showed that both the achieved level of LDL-C and the increase in HDL-C concentration during statin treatment were significant independent predictors for coronary atheroma progression as assessed by coronary intravascular ultrasound. In conclusion, evidence suggests that low levels of HDL-C are associated with an increased CV risk even when LDL-C is reduced to below 1.7 mmol/L (70 mg/dL) with a statin. Moreover, there is mounting evidence that increasing the level of HDL-C has the capacity to reduce CV risk. Thus, there is a compelling case for targeting both the LDL and HDL fractions to reduce CV risk in people with dyslipidemia, high CV risk and low levels of HDL-C. © 2011 Elsevier Ireland Ltd.


Summers F.A.,The Heart Research Institute | Forsman Quigley A.,The Heart Research Institute | Hawkins C.L.,The Heart Research Institute | Hawkins C.L.,University of Sydney
Biochemical and Biophysical Research Communications | Year: 2012

Hypochlorous acid (HOCl) is a potent oxidant produced by the enzyme myeloperoxidase, which is released by neutrophils under inflammatory conditions. Although important in the immune system, HOCl can also damage host tissue, which contributes to the development of disease. HOCl reacts readily with free amino groups to form N-chloramines, which also cause damage in vivo, owing to the extracellular release of myeloperoxidase and production of HOCl. HOCl and N-chloramines react readily with cellular thiols, which causes dysfunction via enzyme inactivation and modulation of redox signaling processes. In this study, the ability of HOCl and model N-chloramines produced on histamine and ammonia at inflammatory sites, to oxidize specific thiol-containing proteins in human coronary artery endothelial cells was investigated. Using a proteomics approach with the thiol-specific probe, 5-iodoacetamidofluorescein, we show that several proteins including peptidylprolyl isomerase A (cyclophilin A), protein disulfide isomerase, glyceraldehyde-3-phosphate dehydrogenase and galectin-1 are particularly sensitive to oxidation by HOCl and N-chloramines formed at inflammatory sites. This will contribute to cellular dysfunction and may play a role in inflammatory disease pathogenesis. © 2012 Elsevier Inc.


Barter P.J.,The Heart Research Institute
Diabetes and Metabolism Journal | Year: 2011

Type 2 diabetes is commonly accompanied by a low level of high density lipoprotein cholesterol (HDL-C) that contributes to the increased cardiovascular risk associated with this condition. Given that HDLs have the ability to improve increase the uptake of glucose by skeletal muscle and to stimulate the secretion of insulin from pancreatic beta cells the possibility arises that a low HDL concentration in type 2 diabetes may also contribute to a worsening of diabetic control. Thus, there is a double case for raising the level of HDL-C in patients with type 2 diabetes: to reduce cardiovascular risk and to improve glycemic control. Approaches to raising HDL-C include lifestyle factors such as weight reduction, increased physical activity and stopping smoking. Of currently available drugs, the most effective is niacin. Newer formulations of niacin are reasonably well tolerated and have the ability to increase HDL-C by up to 30%. The effect of niacin on cardiovascular events in type 2 diabetes is currently being tested in a largescale clinical outcome trial. © 2011 Korean Diabetes Association.


Tso C.,The Heart Research Institute
PloS one | Year: 2012

Blood monocytes are known to express endothelial-like genes during co-culture with endothelium. In this study, the time-dependent change in the phenotype pattern of primary blood monocytes after adhering to endothelium is reported using a novel HLA-A2 mistyped co-culture model. Freshly isolated human PBMCs were co-cultured with human umbilical vein endothelial cells or human coronary arterial endothelial cells of converse human leukocyte antigen A2 (HLA-A2) status. This allows the tracking of the PBMC-derived cells by HLA-A2 expression and assessment of their phenotype pattern over time. PBMCs that adhered to the endothelium at the start of the co-culture were predominantly CD11b+ blood monocytes. After 24 to 72 hours in co-culture, the endothelium-adherent monocytes acquired endothelial-like properties including the expression of endothelial nitric oxide synthase, CD105, CD144 and vascular endothelial growth factor receptor 2. The expression of monocyte/macrophage lineage antigens CD14, CD11b and CD36 were down regulated concomitantly. The adherent monocytes did not express CD115 after 1 day of co-culture. By day 6, the monocyte-derived cells expressed vascular cell adhesion molecule 1 in response to tumour necrosis factor alpha. Up to 10% of the PBMCs adhered to the endothelium. These monocyte-derived cells contributed up to 30% of the co-cultured cell layer and this was dose-dependent on the PBMC seeding density. Human blood monocytes undergo rapid phenotype change to resemble endothelial cells after adhering to endothelium.


Dunn L.,The Heart Research Institute
Journal of visualized experiments : JoVE | Year: 2013

Wound healing and repair are the most complex biological processes that occur in human life. After injury, multiple biological pathways become activated. Impaired wound healing, which occurs in diabetic patients for example, can lead to severe unfavorable outcomes such as amputation. There is, therefore, an increasing impetus to develop novel agents that promote wound repair. The testing of these has been limited to large animal models such as swine, which are often impractical. Mice represent the ideal preclinical model, as they are economical and amenable to genetic manipulation, which allows for mechanistic investigation. However, wound healing in a mouse is fundamentally different to that of humans as it primarily occurs via contraction. Our murine model overcomes this by incorporating a splint around the wound. By splinting the wound, the repair process is then dependent on epithelialization, cellular proliferation and angiogenesis, which closely mirror the biological processes of human wound healing. Whilst requiring consistency and care, this murine model does not involve complicated surgical techniques and allows for the robust testing of promising agents that may, for example, promote angiogenesis or inhibit inflammation. Furthermore, each mouse acts as its own control as two wounds are prepared, enabling the application of both the test compound and the vehicle control on the same animal. In conclusion, we demonstrate a practical, easy-to-learn, and robust model of wound healing, which is comparable to that of humans.

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