Melbourne Medical School Collected Works - Research Publications
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ItemCytotoxic lymphocytes and atherosclerosis: significance, mechanisms and therapeutic challenges(WILEY, 2017-11-01)Cytotoxic lymphocytes encompass natural killer lymphocytes (cells) and cytotoxic T cells that include CD8+ T cells, natural killer (NK) T cells, γ, δ (γδ)-T cells and human CD4 + CD28- T cells. These cells play critical roles in inflammatory diseases and in controlling cancers and infections. Cytotoxic lymphocytes can be activated via a number of mechanisms that may involve dendritic cells, macrophages, cytokines or surface proteins on stressed cells. Upon activation, they secrete pro-inflammatory cytokines as well as anti-inflammatory cytokines, chemokines and cytotoxins to promote inflammation and the development of atherosclerotic lesions including vulnerable lesions, which are strongly implicated in myocardial infarctions and strokes. Here, we review the mechanisms that activate and regulate cytotoxic lymphocyte activity, including activating and inhibitory receptors, cytokines, chemokine receptors-chemokine systems utilized to home to inflamed lesions and cytotoxins and cytokines through which they affect other cells within lesions. We also examine their roles in human and mouse models of atherosclerosis and the mechanisms by which they exert their pathogenic effects. Finally, we discuss strategies for therapeutically targeting these cells to prevent the development of atherosclerotic lesions and vulnerable plaques and the challenge of developing highly targeted therapies that only minimally affect the body's immune system, avoiding the complications, such as increased susceptibility to infections, which are currently associated with many immunotherapies for autoimmune diseases. LINKED ARTICLES: This article is part of a themed section on Targeting Inflammation to Reduce Cardiovascular Disease Risk. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.22/issuetoc and http://onlinelibrary.wiley.com/doi/10.1111/bcp.v82.4/issuetoc.
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ItemShear-sensitive nanocapsule drug release for site-specific inhibition of occlusive thrombus formation(WILEY, 2017-05-01)Essentials Vessel stenosis due to large thrombus formation increases local shear 1-2 orders of magnitude. High shear at stenotic sites was exploited to trigger eptifibatide release from nanocapsules. Local delivery of eptifibatide prevented vessel occlusion without increased tail bleeding times. Local nanocapsule delivery of eptifibatide may be safer than systemic antiplatelet therapies. SUMMARY: Background Myocardial infarction and stroke remain the leading causes of mortality and morbidity. The major limitation of current antiplatelet therapy is that the effective concentrations are limited because of bleeding complications. Targeted delivery of antiplatelet drug to sites of thrombosis would overcome these limitations. Objectives Here, we have exploited a key biomechanical feature specific to thrombosis, i.e. significantly increased blood shear stress resulting from a reduction in the lumen of the vessel, to achieve site-directed delivery of the clinically used antiplatelet agent eptifibatide by using shear-sensitive phosphatidylcholine (PC)-based nanocapsules. Methods PC-based nanocapsules (2.8 × 1012 ) with high-dose encapsulated eptifibatide were introduced into microfluidic blood perfusion assays and into in vivo models of thrombosis and tail bleeding. Results Shear-triggered nanocapsule delivery of eptifibatide inhibited in vitro thrombus formation selectively under stenotic and high shear flow conditions above a shear rate of 1000 s-1 while leaving thrombus formation under physiologic shear rates unaffected. Thrombosis was effectively prevented in in vivo models of vessel wall damage. Importantly, mice infused with shear-sensitive antiplatelet nanocapsules did not show prolonged bleeding times. Conclusions Targeted delivery of eptifibatide by shear-sensitive nanocapsules offers site-specific antiplatelet potential, and may form a basis for developing more potent and safer antiplatelet drugs.
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ItemUrine proteome analysis as a discovery tool in patients with deep vein thrombosis and pulmonary embolism(WILEY-V C H VERLAG GMBH, 2016-05-01)PURPOSE: Early and accurate detection of deep vein thrombosis (DVT) is an important clinical need. Based on the hypothesis that urinary peptides may hold information on DVT in conjunction with pulmonary embolism (PE), the study was aimed at identifying such peptide biomarkers using capillary electrophoresis coupled mass spectrometry. EXPERIMENTAL DESIGN: Patients with symptoms of unprovoked/idiopathic DVT and/or PE were examined by doppler-sonography or angio-computed tomography. Urinary proteome analysis allowed for identification of respective peptide biomarkers. To confirm their biological relevance, we induced PE in mice and assessed human ex vivo thrombi. RESULTS: We identified 62 urinary peptides as DVT-specific biomarkers, i.e. fragments of collagen type I and a fragment of fibrinogen β-chain. The presence of fibrinogen α/β in the acute thrombus, and collagen type I and osteopontin in the older, organized thrombus was demonstrated. The classifier DVT62 established through support vector machine (SVM) modeling based on the 62 identified peptides was validated in an independent cohort of 47 subjects (six cases and 41 controls) with a sensitivity of 100% and specificity of 83%. CONCLUSIONS AND CLINICAL RELEVANCE: Urine proteome analysis enabled the detection of DVT-specific peptides, which were validated in human and mouse tissue. Furthermore, it allowed for the establishment of an urinary-proteome based classifier that is relatively specific for DVT. The data provide the basis for assessment of these biomarkers in a prospective clinical study.
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ItemThe bidirectional interaction between the sympathetic nervous system and immune mechanisms in the pathogenesis of hypertension(WILEY, 2019-06-01)Over the last few years, evidence has accumulated to suggest that hypertension is, at least in part, an immune-mediated inflammatory disorder. Many links between immunity and hypertension have been established and provide a complex framework of mechanistic interactions contributing to the rise in BP. These include immune-mediated inflammatory processes affecting regulatory brain nuclei and interactions with other mediators of cardiovascular regulation such as the sympathetic nervous system. Sympathoexcitation differentially regulates T-cells based upon activation status of the immune cell as well as the resident organ. Exogenous and endogenous triggers activate signalling pathways in innate and adaptive immune cells resulting in pro-inflammatory cytokine production and activation of T-lymphocytes in the cardiovascular and renal regions, now considered major factors in the development of essential hypertension. The inflammatory cascade is sustained and exacerbated by the immune flow via the brain-bone marrow-spleen-gastrointestinal axis and thereby further aggravating immune-mediated pathways resulting in a vicious cycle of established hypertension and target organ damage. This review summarizes the evidence and recent advances in linking immune-mediated inflammation, sympathetic activation and their bidirectional interactions with the development of hypertension. LINKED ARTICLES: This article is part of a themed section on Immune Targets in Hypertension. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.12/issuetoc.
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ItemThe pulmonary microvasculature entraps induced vascular progenitor cells (iVPCs) systemically delivered after cardiac ischemia-reperfusion injury: Indication for preservation of heart function via paracrine effects beyond engraftment(WILEY, 2019-02-01)OBJECTIVE: Stem cell-based regenerative therapies have been intensively studied with the aim to define an ideal cell type for the treatment of myocardial infarction. We tested systemically delivered, platelet-targeted induced vascular progenitor cells (iVPCs) to study their potential to salvage damaged myocardium after ischemia-reperfusion injury. METHODS: Using a mouse model of ischemia-reperfusion injury, we tested the potential of platelet-targeted iVPCs (1 × 106 targ-iVPCs) compared to non-targ-iVPCs and a saline control. Bioluminescence imaging, echocardiography, and histological analyses were performed. RESULTS: Four weeks after ischemia-reperfusion injury, systemic delivery of targ-iVPCs led to reduced fibrosis and infarct size (PBS: 25.7 ± 3.9 vs targ-iVPC: 18.4 ± 6.6 vs non-targ-iVPC: 25.1 ± 3.7%I/LV, P < 0.05), increased neovascularization, and restored cardiac function (PBS: 44.0 ± 4.2 vs targ-iVPC: 54.3 ± 4.5 vs non-targ-iVPC: 46.4 ± 3.8%EF, P < 0.01). Cell tracking experiments revealed entrapment of intravenously injected iVPCs in the pulmonary microvasculature in both cell-treated groups. CONCLUSIONS: Systemic delivery of iVPCs after cardiac ischemia-reperfusion injury is limited by pulmonary entrapment of the cells. Nevertheless, targ-iVPCs reduced infarct size, fibrosis, increased neovascularization, and most importantly retained cardiac function. These findings contribute to the mechanistic discussion of cell-based therapy and ultimately identify activated platelet-targeted iVPCs as candidates for cell therapy and also describe cell therapy benefits without the necessity of engrafting.
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ItemInflammation in acute coronary syndrome: Expression of TLR2 mRNA is increased in platelets of patients with ACS(PUBLIC LIBRARY SCIENCE, 2019-10-23)BACKGROUND: Platelets are key components in atherogenesis and determine the course of its clinical sequelae acute coronary syndrome (ACS). Components of the innate immune system-the superfamily of TLR receptors-are present in platelets and represent a link between atherothrombosis and inflammation. We hypothesize that alteration in platelet TLR mRNA expression is a result of inflammation driving coronary atherosclerosis and may represent an alternative platelet activation pathway in ACS. TLR2-, TLR4- and TLR9- mRNA-expression was determined in ACS patients and compared to patients with invasive exclusion of atherosclerotic lesions of coronary arteries. METHODS: A total of fifty-four patients were enrolled in this clinical retrospective cohort single centre study. Total RNA from sepharose-filtered highly purified platelets was isolated using acid guanidinium thiocyanate-phenol-chloroform extraction and transcribed to cDNA using a first strand cDNA synthesis kit. To determine absolute copy numbers of TLR2, TLR4 and TLR9 we used plasmid based quantitative PCR with normalisation to an internal control. RESULTS: We found that mRNA expression levels of TLR2 but not TLR 4 and 9 are up-regulated in platelets of patients with ACS when compared to patients without coronary atherosclerosis. CONCLUSION: Our results suggest elevated TLR2 mRNA expression in platelets as a biomarker reflecting the underlying inflammation in ACS and possibly severity of coronary atherosclerosis. Platelet TLR2 may represent a link between inflammation and atherothrombosis in ACS.
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ItemNo Preview AvailableGenetic transfer of fusion proteins effectively inhibits VCAM-1-mediated cell adhesion and transmigration via inhibition of cytoskeletal anchorage(WILEY, 2010-01-01)The adhesion of leukocytes to endothelium plays a central role in the development of atherosclerosis and thus represents an attractive therapeutic target for anti-atherosclerotic therapies. Vascular cell adhesion molecule-1 (VCAM-1) mediates both the initial tethering and the firm adhesion of leukocytes to endothelial cells. Our work evaluates the feasibility of using the cytoskeletal anchorage of VCAM-1 as a target for gene therapy. As a proof of concept, integrin alphaIIbbeta3-mediated cell adhesion with clearly defined cytoskeletal anchorage was tested. We constructed fusion proteins containing the intracellular domain of beta3 placed at various distances to the cell membrane. Using cell adhesion assays and immunofluorescence, we established fusion constructs with competitive and dominant negative inhibition of cell adhesion. With the goal being the transfer of the dominant negative mechanism towards VCAM-1 inhibition, we constructed a fusion molecule containing the cytoplasmic domain of VCAM-1. Indeed, VCAM-1 mediated leukocyte adhesion can be inhibited via transfection of DNA encoding the designed VCAM-1 fusion protein. This is demonstrated in adhesion assays under static and flow conditions using CHO cells expressing recombinant VCAM-1 as well as activated endothelial cells. Thus, we are able to describe a novel approach for dominant negative inhibition of leukocyte adhesion to endothelial cells. This approach warrants further development as a novel gene therapeutic strategy that aims for a locally restricted effect at atherosclerotic areas of the vasculature.
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ItemNo Preview AvailableThrombus-Targeted Theranostic Microbubbles: A New Technology towards Concurrent Rapid Ultrasound Diagnosis and Bleeding-free Fibrinolytic Treatment of Thrombosis(IVYSPRING INT PUBL, 2016-01-01)RATIONALE: Myocardial infarction and stroke are leading causes of morbidity/mortality. The typical underlying pathology is the formation of thrombi/emboli and subsequent vessel occlusion. Systemically administered fibrinolytic drugs are the most effective pharmacological therapy. However, bleeding complications are relatively common and this risk as such limits their broader use. Furthermore, a rapid non-invasive imaging technology is not available. Thereby, many thrombotic events are missed or only diagnosed when ischemic damage has already occurred. OBJECTIVE: Design and preclinical testing of a novel 'theranostic' technology for the rapid non-invasive diagnosis and effective, bleeding-free treatment of thrombosis. METHODS AND RESULTS: A newly created, innovative theranostic microbubble combines a recombinant fibrinolytic drug, an echo-enhancing microbubble and a recombinant thrombus-targeting device in form of an activated-platelet-specific single-chain antibody. After initial in vitro proof of functionality, we tested this theranostic microbubble both in ultrasound imaging and thrombolytic therapy using a mouse model of ferric-chloride-induced thrombosis in the carotid artery. We demonstrate the reliable highly sensitive detection of in vivo thrombi and the ability to monitor their size changes in real time. Furthermore, these theranostic microbubbles proofed to be as effective in thrombolysis as commercial urokinase but without the prolongation of bleeding time as seen with urokinase. CONCLUSIONS: We describe a novel theranostic technology enabling simultaneous diagnosis and treatment of thrombosis, as well as monitoring of success or failure of thrombolysis. This technology holds promise for major progress in rapid diagnosis and bleeding-free thrombolysis thereby potentially preventing the often devastating consequences of thrombotic disease in many patients.
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ItemMicroparticles: major transport vehicles for distinct microRNAs in circulation(OXFORD UNIV PRESS, 2012-03-15)AIMS: Circulating microRNAs (miRNAs) have attracted major interest as biomarkers for cardiovascular diseases. Since RNases are abundant in circulating blood, there needs to be a mechanism protecting miRNAs from degradation. We hypothesized that microparticles (MP) represent protective transport vehicles for miRNAs and that these are specifically packaged by their maternal cells. METHODS AND RESULTS: Conventional plasma preparations, such as the ones used for biomarker detection, are shown to contain substantial numbers of platelet-, leucocyte-, and endothelial cell-derived MP. To analyse the widest spectrum of miRNAs, Next Generation Sequencing was used to assess miRNA profiles of MP and their corresponding stimulated and non-stimulated cells of origin. THP-1 (monocytic origin) and human umbilical vein endothelial cell (HUVEC) MP were used for representing circulating MP at a high purity. miRNA profiles of MP differed significantly from those of stimulated and non-stimulated maternal THP-1 cells and HUVECs, respectively. Quantitative reverse transcription-polymerase chain reaction of miRNAs which have been associated with cardiovascular diseases also demonstrated significant differences in miRNA profiles between platelets and their MP. Notably, the main fraction of miRNA in plasma was localized in MP. Furthermore, miRNA profiles of MP differed significantly between patients with stable and unstable coronary artery disease. CONCLUSION: Circulating MP represent transport vehicles for large numbers of specific miRNAs, which have been associated with cardiovascular diseases. miRNA profiles of MP are significantly different from their maternal cells, indicating an active mechanism of selective 'packaging' from cells into MP. These findings describe an interesting mechanism for transferring gene-regulatory function from MP-releasing cells to target cells via MP circulating in blood.
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