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Kockx M.,ANZAC Research Institute | Glaros E.,University of Sydney | Glaros E.,Center for Vascular Research | Ng T.W.,University of New South Wales | And 12 more authors.
Arteriosclerosis, Thrombosis, and Vascular Biology | Year: 2016

Objective-Cyclosporin A (CsA) is an immunosuppressant commonly used to prevent organ rejection but is associated with hyperlipidemia and an increased risk of cardiovascular disease. Although studies suggest that CsA-induced hyperlipidemia is mediated by inhibition of low-density lipoprotein receptor (LDLr)-mediated lipoprotein clearance, the data supporting this are inconclusive. We therefore sought to investigate the role of the LDLr in CsA-induced hyperlipidemia by using Ldlr-knockout mice (Ldlr-/-). Approach and Results-Ldlr-/- and wild-Type (wt) C57Bl/6 mice were treated with 20 mg/kg per d CsA for 4 weeks. On a chow diet, CsA caused marked dyslipidemia in Ldlr-/- but not in wt mice. Hyperlipidemia was characterized by a prominent increase in plasma very low-density lipoprotein and intermediate-density lipoprotein/LDL with unchanged plasma high-density lipoprotein levels, thus mimicking the dyslipidemic profile observed in humans. Analysis of specific lipid species by liquid chromatography-Tandem mass spectrometry suggested a predominant effect of CsA on increased very low-density lipoprotein-IDL/LDL lipoprotein number rather than composition. Mechanistic studies indicated that CsA did not alter hepatic lipoprotein production but did inhibit plasma clearance and hepatic uptake of [14C]cholesteryl oleate and glycerol tri[3H]oleate-double-labeled very low-density lipoprotein-like particles. Further studies showed that CsA inhibited plasma lipoprotein lipase activity and increased levels of apolipoprotein C-III and proprotein convertase subtilisin/kexin type 9. Conclusions-We demonstrate that CsA does not cause hyperlipidemia via direct effects on the LDLr. Rather, LDLr deficiency plays an important permissive role for CsA-induced hyperlipidemia, which is associated with abnormal lipoprotein clearance, decreased lipoprotein lipase activity, and increased levels of apolipoprotein C-III and proprotein convertase subtilisin/kexin type 9. Enhancing LDLr and lipoprotein lipase activity and decreasing apolipoprotein C-III and proprotein convertase subtilisin/kexin type 9 levels may therefore provide attractive treatment targets for patients with hyperlipidemia receiving CsA. © 2016 American Heart Association, Inc.


Cai H.,University of New South Wales | Santiago F.S.,University of New South Wales | Prado-Lourenco L.,University of New South Wales | Wang B.,University of New South Wales | And 17 more authors.
Science Translational Medicine | Year: 2012

Worldwide, one in three cancers is skin-related, with increasing incidence in many populations. Here, we demonstrate the capacity of a DNAzyme-targeting c-jun mRNA, Dz13, to inhibit growth of two common skin cancer types - basal cell and squamous cell carcinomas - in a therapeutic setting with established tumors. Dz13 inhibited tumor growth in both immunodeficient and immunocompetent syngeneic mice and reduced lung nodule formation in a model of metastasis. In addition, Dz13 suppressed neovascularization in tumor-bearing mice and zebrafish and increased apoptosis of tumor cells. Dz13 inhibition of tumor growth, which required an intact catalytic domain, was due in part to the induction of tumor immunity. In a series of good laboratory practice-compliant toxicology studies in cynomolgus monkeys, minipigs, and rodents, the DNAzyme was found to be safe and well tolerated. It also did not interfere in more than 70 physiologically relevant in vitro bioassays, suggesting a reduced propensity for off-target effects. If these findings hold true in clinical trials, Dz13 may provide a safe, effective therapy for human skin cancer.


News Article | December 12, 2016
Site: www.eurekalert.org

An important parcel must be delivered to the correct place. It is so important that it can be a question of life or death. However, uneven streets and missing railings risk to bring it off the road and leave it undelivered. This is not the trailer of a drama, but what happens to anti-cancer drugs traveling towards the tumor area, but unable to reach it because of dysfunctional blood vessels. The Center for Vascular Research, within the Institute for Basic Science (IBS) discovered that their antisepsis antibody ABTAA (Ang2-Binding and Tie2-Activating Antibody) also reduces tumor volume and improves the delivery of anti-cancer drugs. Published in Cancer Cell, this study demonstrates that ABTAA restores the structural and functional integrity of tumor blood vessels in three different tumor models: breast, lungs and brain. Blood vessels inside and around an established tumor can be described as a chaotic and dysfunctional labyrinth. While the inner walls of healthy blood vessels are surrounded and supported by endothelial cells and other cells called pericytes; in the established tumor, the endothelial junctions are broken apart and pericytes are also detached. Blood flow into and from the tumor is severely retarded and tumor vessels lacking an intact vessel wall become leaky. This microenvironment causes limited drug delivery to the tumor and leads to inadequate oxygen supply (hypoxia) and even metastasis. IBS scientists found that the antibody ABTAA normalizes the tumor vessels and hence, change the whole tumor microenvironment. "We call it normalization of tumor vessels, because it resembles closely the wall architecture of healthy, normal vessels," explains PARK Jin-Sung, first author of the study. And continues: "Tumor can adapt to hypoxia and get more aggressive, so we tried to prevent this transition by normalizing tumor vessels. ABTAA changes the whole tumor environment, oxygenation status and level of lactate, so that the immune cells and drugs can reach the core regions of the tumor more easily. In this way, we create a favorable ground for tumor treatment." In an attempt to generate antibodies targeting the protein Ang2, which is specifically expressed by endothelial cells in stressful conditions like in tumor, the team unexpectedly discovered that ABTAA has a peculiar way of working and a dual function. ABTAA indeed not only blocks Ang2, but it is also able to activate Tie2 at the same time. Tie2 is a receptor present on the cell membrane of endothelial cells. ABTAA causes Ang2 to cluster together and to strongly activate Tie2 receptors. "If we activate Tie2, we can efficiently normalize tumor vessels, enhance drug delivery and change the whole microenvironment," explains KOH Gou Young, Director of the Center for Vascular Research. Several pharmaceutical companies are developing Ang2-blocking antibodies to cure cancer. However, even if these antibodies significantly inhibit tumor progression, they do not stop tumor hypoxia. Moreover, most of the anti-cancer drugs target the tumor at its early stage, when tumors are still hard to diagnose. ABTAA, instead, works with tumors that are already rooted: "When the tumor is established, hypoxia is the main driver of tumor progression. So, if we eliminate hypoxia, we make the tumor more mild, by reducing its progression and metastasis," comments Koh. IBS researchers tested ABTAA in mice with three different types of tumors that show high levels of Ang2: glioma (a type a brain tumor), lung carcinoma and breast cancer. They also compared the effect of ABTAA with ABA, another antibody that blocks Ang2 but misses the Tie2 activating properties. In all three cases, ABTAA was superior to ABA in inducing tumor vessel normalization, which led to a better delivery of the anti-cancer drugs into the tumor core region. Glioma is one of the so-called intractable disease, because of its poor prognosis and treatment. IBS scientists found that the glioma volume was reduced 39% by ABTAA and 17% by ABA. ABTAA profoundly reduced vascular leakage and edema formation in glioma through promoting vascular tightening. Moreover, when ABTAA was administered together with the chemotherapeutic drug temozolomide (TMZ), the tumor volume reduces further (76% by ABTAA+TMZ, 51% by ABA+TMZ, and 36% by TMZ). In the Lewis Lung Carcinoma (LLC) tumor model, the research team administered ABTAA together with a chemotherapeutic drug called cisplatin (Cpt) and observed a greater suppression of tumor growth (52%) compared with the controls and increased overall survival. Moreover, ABTAA+Cpt led to a marked increase in necrotic area within tumors. Finally, in a spontaneous breast cancer model, ABTAA delayed tumor growth and enhanced the anti-tumor effect of Cpt. In the future, the team would like to further understand the underlying relationship between faulty blood vessels and diseases. "We would like to apply this antibody to an organ that is rich in blood vessels, that is the eye, and see if this antibody can be useful to treat eye diseases such as age-related macular degeneration and diabetic retinopathy," concludes Koh.


Lonn M.E.,Center for Vascular Research | Lonn M.E.,University of Sydney | Dennis J.M.,Center for Vascular Research | Dennis J.M.,University of Sydney | Stocker R.,Victor Chang Cardiac Research Institute
Free Radical Biology and Medicine | Year: 2012

This review addresses the role of oxidative processes in atherosclerosis and its resulting cardiovascular disease by focusing on the outcome of antioxidant interventions. Although there is unambiguous evidence for the presence of heightened oxidative stress and resulting damage in atherosclerosis, it remains to be established whether this represents a cause or a consequence of the disease. This critical question is complicated further by the increasing realization that oxidative processes, including those related to signaling, are part of normal cell function. Overall, the results from animal interventions suggest that antioxidants provide benefit neither generally nor consistently. Where benefit is observed, it appears to be achieved at least in part via modulation of biological processes such as increase in nitric oxide bioavailability and induction of protective enzymes such as heme oxygenase-1, rather than via inhibition of oxidative processes and lipid oxidation in the arterial wall. Exceptions to this may be situations of multiple/excessive stress, the relevance of which for humans is not clear. This interpretation is consistent with the overall disappointing outcome of antioxidant interventions in humans and can be rationalized by the spatial compartmentalization of cellular oxidative signaling and/or damage, complex roles of oxidant-producing enzymes, and the multifactorial nature of atherosclerosis. © 2012 Elsevier Inc. All rights reserved.


Harith H.H.,Center for Vascular Research | Harith H.H.,University of New South Wales | Harith H.H.,University Putra Malaysia | Di Bartolo B.A.,The Heart Research Institute | And 6 more authors.
Journal of Diabetes | Year: 2016

Background: Insulin regulates glucose homeostasis but can also promote vascular smooth muscle (VSMC) proliferation, important in atherogenesis. Recently, we showed that tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) stimulates intimal thickening via accelerated growth of VSMCs. The aim of the present study was to determine whether insulin-induced effects on VSMCs occur via TRAIL. Methods: Expression of TRAIL and TRAIL receptor in response to insulin and glucose was determined by polymerase chain reaction. Transcriptional activity was assessed using wild-type and site-specific mutations of the TRAIL promoter. Chromatin immunoprecipitation studies were performed. VSMC proliferation and apoptosis was measured. Results: Insulin and glucose exposure to VSMC for 24 h stimulated TRAIL mRNA expression. This was also evident at the transcriptional level. Both insulin- and glucose-inducible TRAIL transcriptional activity was blocked by dominant-negative specificity protein-1 (Sp1) overexpression. There are five functional Sp1-binding elements (Sp1-1, Sp1-2, Sp-5/6 and Sp1-7) on the TRAIL promoter. Insulin required the Sp1-1 and Sp1-2 sites, but glucose needed all Sp1-binding sites to induce transcription. Furthermore, insulin (but not glucose) was able to promote VSMC proliferation over time, associated with increased decoy receptor-2 (DcR2) expression. In contrast, chronic 5-day exposure of VSMC to 1 µg/mL insulin repressed TRAIL and DcR2 expression, and reduced Sp1 enrichment on the TRAIL promoter. This was associated with increased cell death. Conclusions: The findings of the present study provide a new mechanistic insight into how TRAIL is regulated by insulin. This may have significant implications at different stages of diabetes-associated cardiovascular disease. Thus, TRAIL may offer a novel therapeutic solution to combat insulin-induced vascular pathologies. © 2015 Ruijin Hospital, Shanghai Jiaotong University School of Medicine and Wiley Publishing Asia Pty Ltd


Collinson E.J.,Center for Vascular Research | Wimmer-Kleikamp S.,Center for Vascular Research | Gerega S.K.,University of Sydney | Yang Y.H.,University of Sydney | And 3 more authors.
Journal of Biological Chemistry | Year: 2011

Heme oxygenase-1 (HO-1) degrades heme and protects cells from oxidative challenge. This antioxidant activity is thought to result from the HO-1 enzymatic activity, manifested by Abstract decrease in the concentration of the pro-oxidant substrate heme, and an increase in the antioxidant product bilirubin. Using a global transcriptional approach, and yeast as a model, we show that HO-1 affords cellular protection via up-regulation of transcripts encoding enzymes involved in cellular antioxidant defense, rather than via its oxygenase activity. Like mammalian cells, yeast responds to oxidative stress by expressing its HO-1 homolog and, compared with the wild type, heme oxygenase-null mutant cells have increased sensitivity toward oxidants that is rescued by overexpression of human HO-1 or its yeast homolog. Increased oxidant sensitivity of heme oxygenase-null mutant cells is explained by a decrease in the expression of the genes encoding γ-glutamylcysteine synthetase, glutathione peroxidase, catalase, and methionine sulfoxide reductase, because overexpression of any of these genes affords partial, and overexpression of all four genes provides complete, protection to the null mutant. Genes encoding antioxidant enzymes represent only a small portion of the 480 differentially expressed transcripts in heme oxygenase-null mutants. Transcriptional regulation may be explained by the nuclear localization of heme oxygenase observed in oxidantchallenged cells. Our results challenge the notion that HO-1 functions simply as a catabolic and antioxidant enzyme. They indicate much broader functions for HO-1, the unraveling of which may help explain the multiple biological responses reported in animals as a result of altered HO-1 expression. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.


Ishii H.,University of New South Wales | Hulett M.D.,La Trobe University | Li J.-M.,University of New South Wales | Santiago F.S.,University of New South Wales | And 2 more authors.
International Journal of Oncology | Year: 2012

The GLI-Krüppel zinc finger factor yin yang-1 (YY1) is a complex protein that regulates a variety of processes including transcription, proliferation, development and differentiation. YY1 inhibits cell growth in a cell typespecific manner. The role played by YY1 in its control of tumor cell growth is unclear and controversial. We show here that YY1 can suppress the growth of different tumor cell types in vitro, including human breast carcinoma cells and glioblastoma cells. YY1 also blocked the growth of 13762 MAT mammary adenocarcinoma isografts in rats. YY1 inhibited 13762 MAT tumor growth by approximately 80% compared with the GFP alone group 21 days after injection. YY1 inhibited proliferating cell nuclear antigen (PCNA) expression and pRb Ser249/Thr252 phosphorylation without influencing tumor microvascular density. Moreover, YY1 inhibited p21 WAF1/Cip1complex formation with cdk4 and cyclin D1. These findings demonstrate that YY1 can negatively regulate the growth of multiple malignant cell types.


PubMed | Center for Vascular Research and The Heart Research Institute
Type: Journal Article | Journal: Journal of diabetes | Year: 2016

Insulin regulates glucose homeostasis but can also promote vascular smooth muscle (VSMC) proliferation, important in atherogenesis. Recently, we showed that tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) stimulates intimal thickening via accelerated growth of VSMCs. The aim of the present study was to determine whether insulin-induced effects on VSMCs occur via TRAIL.Expression of TRAIL and TRAIL receptor in response to insulin and glucose was determined by polymerase chain reaction. Transcriptional activity was assessed using wild-type and site-specific mutations of the TRAIL promoter. Chromatin immunoprecipitation studies were performed. VSMC proliferation and apoptosis was measured.Insulin and glucose exposure to VSMC for 24h stimulated TRAIL mRNA expression. This was also evident at the transcriptional level. Both insulin- and glucose-inducible TRAIL transcriptional activity was blocked by dominant-negative specificity protein-1 (Sp1) overexpression. There are five functional Sp1-binding elements (Sp1-1, Sp1-2, Sp-5/6 and Sp1-7) on the TRAIL promoter. Insulin required the Sp1-1 and Sp1-2 sites, but glucose needed all Sp1-binding sites to induce transcription. Furthermore, insulin (but not glucose) was able to promote VSMC proliferation over time, associated with increased decoy receptor-2 (DcR2) expression. In contrast, chronic 5-day exposure of VSMC to 1g/mL insulin repressed TRAIL and DcR2 expression, and reduced Sp1 enrichment on the TRAIL promoter. This was associated with increased cell death.The findings of the present study provide a new mechanistic insight into how TRAIL is regulated by insulin. This may have significant implications at different stages of diabetes-associated cardiovascular disease. Thus, TRAIL may offer a novel therapeutic solution to combat insulin-induced vascular pathologies.

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