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Plaque neovessel maturation enhancement by VEGFR2 blockade reduces intraplaque haemorrhage
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Abstracts / Atherosclerosis 244 (2016) e1ee12
A NOVEL ASSAY USING SYNTHETIC PROTEIN SCAFFOLDS TO DETECT SOLUBLE LECTIN-LIKE OXIDISED LOW DENSITY LIPOPROTEIN RECEPTOR-1 IN VASCULAR DISEASE J.R. De Siqueira 1, 4, *, R.A. Seese 2, D.C. Tomlinson 1, D.A. Russell 3, 4, R. Ajjan 3, S. Ponnambalam 1, S. Homer-Vanniasinkam 1, 4. 1 School of Molecular & Cellular Biology, University of Leeds, Leeds LS2 9JT, UK; 2 University of Leeds Medical School, University of Leeds, Leeds LS2 9JT, UK; 3 Leeds Institute of Cardiovascular and Metabolic Medicine, University of Leeds, Leeds LS2 9JT, UK; 4 Leeds Vascular Institute, Leeds General Infirmary, Great George Street, Leeds LS1 3EX, UK * Presenting author. Introduction: Lectin-like Oxidised Low Density Lipoprotein (OxLDL) Receptor 1 (LOX-1) mediates uptake of OxLDL particles in vascular tissues and is implicated in atherosclerosis initiation and progression. Fragments of soluble LOX-1 (sLOX-1) are proteolysed and shed into the extracellular medium and act as a biomarker of chronic disease states including type 2 diabetes mellitus, acute coronary syndrome and obesity. Adhirons are synthetic protein scaffolds that can be directed against a wide range of molecular surfaces. We aimed to investigate whether Adhirons directed against human sLOX-1 could be used to monitor disease status. Methods: The bacterium E.coli was used to produce recombinant Adhirons that recognised human sLOX-1. Different ELISA-based assays were analysed for the ability of Adhirons to bind and detect sLOX-1 in biological buffers and human plasma. Results: Hybrid antibody-sLOX-1-Adhiron and homogenous AdhironsLOX-1-Adhiron assays could detect sLOX-1 levels down to picogram per millilitre sensitivity. Analysis of sLOX-1 levels using conventional antibody or Adhiron-based assays suggests that (1) sLOX-1 levels are elevated in chronic disease states, (2) Adhirons have high affinity and specificity for sLOX-1, and (3) Adhiron-based assays are a viable alternative for monitoring sLOX-1 biomarker levels. Conclusions: This study demonstrates that Adhirons can act as probes for monitoring sLOX-1 levels in vascular disease states. Further refinement and optimisation is needed for monitoring sLOX-1 levels in human blood samples and relating this to patient disease status. These studies provide a platform for using sLOX-1 as a biomarker in acute and chronic vascular disease.
PLAQUE NEOVESSEL MATURATION ENHANCEMENT BLOCKADE REDUCES INTRAPLAQUE HAEMORRHAGE
BY
VEGFR2
M.R. de Vries 1, 2, *, H.A.B. Peters 1,2, J.F. Hamming 1, M.J. Goumans 2, 3, P.H.A. Quax 1, 2. 1 Department of Surgery, Leiden University Medical Center, Leiden, The Netherlands; 2 Einthoven Laboratory for Experimental Vascular Medicine, Leiden University Medical Center, Leiden, The Netherlands; 3 Department of Molecular Cell Biology, Leiden University Medical Center, Leiden, The Netherlands * Presenting author. Rationale: Vein graft patency rates are poor due to extensive neointimal thickening, atherosclerosis and lesion instability. We have previously shown that murine atherosclerotic vein grafts exhibit leaky neovessels. VEGF-VEGF Receptor 2 (VEGFR2) and the angiopoietin (Ang)-Tie2 system, are the main regulators of neovessel maturation. We hypothesize that blockade of VEGFR2 induces plaque neovessel maturation resulting in reduced intraplaque haemorrhage and increased plaque stability. Methods: Donor caval veins were engrafted in carotid arteries of recipient hypercholesterolemic ApoE3*Leiden mice (n ¼ 14/group). Mice were treated biweekly with VEGFR2 blocking antibodies (DC101) or control IgG antibodies (10 mg/kg) until sacrifice at day 28.
Results: DC101 treatment resulted in a striking 50% decrease in vein graft segments that expressed intraplaque haemorrhage. This was accompanied by a significant 25-fold decrease in extravasated erythrocytes. Interestingly, destabilizing Ang2 was significantly decreased in the DC101 group whereas stabilizing Ang1 was not affected. Furthermore, endothelial cells seem to acquire a more quiescent state upon VEGFR2 blockade since the expression of Connexin 40 was significantly increased whereas ICAM-1 was down regulated. Vein graft lesion size was significantly reduced in the DC101 group, which was accompanied by a significant reduction in macrophage content and significant increases in smooth muscle cell and collagen content. Conclusions: Blockade of VEGFR2 leads to reduced intraplaque haemorrhage, decreased plaque size and increased plaque stability as a result of increased neovessel maturation. This identifies neovessel maturation as an attractive target for the treatment of unstable atherosclerotic diseases.
NECROX-7 ATTENUATES ATHEROSCLEROSIS AND PLAQUE NECROSIS IN APOE KNOCKOUT MICE M.O.J. Grootaert 1, *, S.H. Kim 2, H. Van Spaendonk 1, G.R.Y. De Meyer 1, D.M. Schrijvers 1, W. Martinet 1. 1 Laboratory of Physiopharmacology, University of Antwerp, Antwerp, Belgium; 2 R&D Park, LG Life Sciences, Ltd., Daejeon, South Korea * Presenting author. A large necrotic core is a key player in atherosclerotic plaque instability. Necrotic cellular debris accumulates in the lipid-rich core and promotes inflammation and ultimately plaque rupture. Although the role of necrosis in atherosclerosis is rather clear-cut, not many strategies have been performed up till now to specifically target plaque necrosis. In this study, we evaluated the anti-atherogenic potential of NecroX-7, a novel necrosis inhibitor with anti-oxidative properties. Male ApoE knockout mice were treated with NecroX-7 (30mg/kg) or vehicle 3x/week via intraperitoneal injections for 16 weeks. Simultaneously, mice were fed a western-type diet to induce plaque formation. NecroX-7 lowered circulating VLDL and LDL cholesterol levels and reduced total lipid burden in the thoracic aorta of ApoE knockout mice when compared to vehicle-treated control mice. Atherosclerotic plaques in the ascending aorta showed a significant decrease in necrosis and total plaque size in NecroX-7-treated ApoE knockout mice as compared to control mice. However, cellular plaque composition was not different between both groups. In vitro, NecroX-7 (10mM) prevented tert-butyl hydroperoxide (tBHP)-induced ROS formation, necrosis and high-mobility group box 1 (HMGB1) release in primary macrophages. Interestingly, NecroX-7 preferentially protected M2 macrophages against tBHP-induced necrosis and reduced iNOS mRNA expression in tBHP-treated M1 macrophages. Furthermore, necroptosis and apoptosis were not affected by NecroX-7. We may conclude that NecroX-7, besides its anti-oxidative and anti-necrotic potential, also exerts anti-inflammatory and cholesterol-lowering effects, and could be a new promising multipotent drug for the treatment of atherosclerosis.
DOES MYELOID EXPRESSION OF TRIB1 REGULATE PLASMA LIPID LEVELS Adrienn Angyal a,1, Zabran Ilyas a,1, Eva Hadadi a, Jessica Johnston a, Mark Ariaans a, Ronald Kraus b, Heather Wilson a, Robert Bauer c, Daniel Rader c, Sheila Francis a, Endre Kiss-Toth a. a Department of Cardiovascular Science, University of Sheffield, Sheffield, UK; b Life Sciences Division, Lawrence Berkeley Laboratory, Berkeley, CA, USA; c Cardiovascular Institute and the Institute for Translational Medicine and Therapeutics, University of Pennsylvania, Philadelphia, USA Aim: TRIB1 is a pseudokinase and recent GWAS analyses have linked this gene to the control of lipid homeostasis. Analysis of full body TRIB1 knockout and a liver specific TRIB1 overexpressing mouse have
1
These authors contributed equally to this work
Abstracts / Atherosclerosis 244 (2016) e1ee12
A NOVEL ASSAY USING SYNTHETIC PROTEIN SCAFFOLDS TO DETECT SOLUBLE LECTIN-LIKE OXIDISED LOW DENSITY LIPOPROTEIN RECEPTOR-1 IN VASCULAR DISEASE J.R. De Siqueira 1, 4, *, R.A. Seese 2, D.C. Tomlinson 1, D.A. Russell 3, 4, R. Ajjan 3, S. Ponnambalam 1, S. Homer-Vanniasinkam 1, 4. 1 School of Molecular & Cellular Biology, University of Leeds, Leeds LS2 9JT, UK; 2 University of Leeds Medical School, University of Leeds, Leeds LS2 9JT, UK; 3 Leeds Institute of Cardiovascular and Metabolic Medicine, University of Leeds, Leeds LS2 9JT, UK; 4 Leeds Vascular Institute, Leeds General Infirmary, Great George Street, Leeds LS1 3EX, UK * Presenting author. Introduction: Lectin-like Oxidised Low Density Lipoprotein (OxLDL) Receptor 1 (LOX-1) mediates uptake of OxLDL particles in vascular tissues and is implicated in atherosclerosis initiation and progression. Fragments of soluble LOX-1 (sLOX-1) are proteolysed and shed into the extracellular medium and act as a biomarker of chronic disease states including type 2 diabetes mellitus, acute coronary syndrome and obesity. Adhirons are synthetic protein scaffolds that can be directed against a wide range of molecular surfaces. We aimed to investigate whether Adhirons directed against human sLOX-1 could be used to monitor disease status. Methods: The bacterium E.coli was used to produce recombinant Adhirons that recognised human sLOX-1. Different ELISA-based assays were analysed for the ability of Adhirons to bind and detect sLOX-1 in biological buffers and human plasma. Results: Hybrid antibody-sLOX-1-Adhiron and homogenous AdhironsLOX-1-Adhiron assays could detect sLOX-1 levels down to picogram per millilitre sensitivity. Analysis of sLOX-1 levels using conventional antibody or Adhiron-based assays suggests that (1) sLOX-1 levels are elevated in chronic disease states, (2) Adhirons have high affinity and specificity for sLOX-1, and (3) Adhiron-based assays are a viable alternative for monitoring sLOX-1 biomarker levels. Conclusions: This study demonstrates that Adhirons can act as probes for monitoring sLOX-1 levels in vascular disease states. Further refinement and optimisation is needed for monitoring sLOX-1 levels in human blood samples and relating this to patient disease status. These studies provide a platform for using sLOX-1 as a biomarker in acute and chronic vascular disease.
PLAQUE NEOVESSEL MATURATION ENHANCEMENT BLOCKADE REDUCES INTRAPLAQUE HAEMORRHAGE
BY
VEGFR2
M.R. de Vries 1, 2, *, H.A.B. Peters 1,2, J.F. Hamming 1, M.J. Goumans 2, 3, P.H.A. Quax 1, 2. 1 Department of Surgery, Leiden University Medical Center, Leiden, The Netherlands; 2 Einthoven Laboratory for Experimental Vascular Medicine, Leiden University Medical Center, Leiden, The Netherlands; 3 Department of Molecular Cell Biology, Leiden University Medical Center, Leiden, The Netherlands * Presenting author. Rationale: Vein graft patency rates are poor due to extensive neointimal thickening, atherosclerosis and lesion instability. We have previously shown that murine atherosclerotic vein grafts exhibit leaky neovessels. VEGF-VEGF Receptor 2 (VEGFR2) and the angiopoietin (Ang)-Tie2 system, are the main regulators of neovessel maturation. We hypothesize that blockade of VEGFR2 induces plaque neovessel maturation resulting in reduced intraplaque haemorrhage and increased plaque stability. Methods: Donor caval veins were engrafted in carotid arteries of recipient hypercholesterolemic ApoE3*Leiden mice (n ¼ 14/group). Mice were treated biweekly with VEGFR2 blocking antibodies (DC101) or control IgG antibodies (10 mg/kg) until sacrifice at day 28.
Results: DC101 treatment resulted in a striking 50% decrease in vein graft segments that expressed intraplaque haemorrhage. This was accompanied by a significant 25-fold decrease in extravasated erythrocytes. Interestingly, destabilizing Ang2 was significantly decreased in the DC101 group whereas stabilizing Ang1 was not affected. Furthermore, endothelial cells seem to acquire a more quiescent state upon VEGFR2 blockade since the expression of Connexin 40 was significantly increased whereas ICAM-1 was down regulated. Vein graft lesion size was significantly reduced in the DC101 group, which was accompanied by a significant reduction in macrophage content and significant increases in smooth muscle cell and collagen content. Conclusions: Blockade of VEGFR2 leads to reduced intraplaque haemorrhage, decreased plaque size and increased plaque stability as a result of increased neovessel maturation. This identifies neovessel maturation as an attractive target for the treatment of unstable atherosclerotic diseases.
NECROX-7 ATTENUATES ATHEROSCLEROSIS AND PLAQUE NECROSIS IN APOE KNOCKOUT MICE M.O.J. Grootaert 1, *, S.H. Kim 2, H. Van Spaendonk 1, G.R.Y. De Meyer 1, D.M. Schrijvers 1, W. Martinet 1. 1 Laboratory of Physiopharmacology, University of Antwerp, Antwerp, Belgium; 2 R&D Park, LG Life Sciences, Ltd., Daejeon, South Korea * Presenting author. A large necrotic core is a key player in atherosclerotic plaque instability. Necrotic cellular debris accumulates in the lipid-rich core and promotes inflammation and ultimately plaque rupture. Although the role of necrosis in atherosclerosis is rather clear-cut, not many strategies have been performed up till now to specifically target plaque necrosis. In this study, we evaluated the anti-atherogenic potential of NecroX-7, a novel necrosis inhibitor with anti-oxidative properties. Male ApoE knockout mice were treated with NecroX-7 (30mg/kg) or vehicle 3x/week via intraperitoneal injections for 16 weeks. Simultaneously, mice were fed a western-type diet to induce plaque formation. NecroX-7 lowered circulating VLDL and LDL cholesterol levels and reduced total lipid burden in the thoracic aorta of ApoE knockout mice when compared to vehicle-treated control mice. Atherosclerotic plaques in the ascending aorta showed a significant decrease in necrosis and total plaque size in NecroX-7-treated ApoE knockout mice as compared to control mice. However, cellular plaque composition was not different between both groups. In vitro, NecroX-7 (10mM) prevented tert-butyl hydroperoxide (tBHP)-induced ROS formation, necrosis and high-mobility group box 1 (HMGB1) release in primary macrophages. Interestingly, NecroX-7 preferentially protected M2 macrophages against tBHP-induced necrosis and reduced iNOS mRNA expression in tBHP-treated M1 macrophages. Furthermore, necroptosis and apoptosis were not affected by NecroX-7. We may conclude that NecroX-7, besides its anti-oxidative and anti-necrotic potential, also exerts anti-inflammatory and cholesterol-lowering effects, and could be a new promising multipotent drug for the treatment of atherosclerosis.
DOES MYELOID EXPRESSION OF TRIB1 REGULATE PLASMA LIPID LEVELS Adrienn Angyal a,1, Zabran Ilyas a,1, Eva Hadadi a, Jessica Johnston a, Mark Ariaans a, Ronald Kraus b, Heather Wilson a, Robert Bauer c, Daniel Rader c, Sheila Francis a, Endre Kiss-Toth a. a Department of Cardiovascular Science, University of Sheffield, Sheffield, UK; b Life Sciences Division, Lawrence Berkeley Laboratory, Berkeley, CA, USA; c Cardiovascular Institute and the Institute for Translational Medicine and Therapeutics, University of Pennsylvania, Philadelphia, USA Aim: TRIB1 is a pseudokinase and recent GWAS analyses have linked this gene to the control of lipid homeostasis. Analysis of full body TRIB1 knockout and a liver specific TRIB1 overexpressing mouse have
1
These authors contributed equally to this work