Melbourne Medical School Collected Works - Research Publications

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    The 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
    Ziegler, M ; Haigh, K ; Thao, N ; Wang, X ; Lim, B ; Yap, ML ; Eddy, EM ; Haigh, JJ ; Peter, K (WILEY, 2019-02)
    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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    Thrombus-Targeted Theranostic Microbubbles: A New Technology towards Concurrent Rapid Ultrasound Diagnosis and Bleeding-free Fibrinolytic Treatment of Thrombosis
    Wang, X ; Gkanatsas, Y ; Palasubramaniam, J ; Hohmann, JD ; Chen, YC ; Lim, B ; Hagemeyer, CE ; Peter, K (IVYSPRING INT PUBL, 2016)
    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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    A Unique Recombinant Fluoroprobe Targeting Activated Platelets Allows In Vivo Detection of Arterial Thrombosis and Pulmonary Embolism Using a Novel Three-Dimensional Fluorescence Emission Computed Tomography (FLECT) Technology
    Lim, B ; Yao, Y ; Huang, AL-I ; Yap, ML ; Flierl, U ; Palasubramaniam, J ; Zaldivia, MTK ; Wang, X ; Peter, K (IVYSPRING INT PUBL, 2017)
    Progress in pharmaceutical development is highly-dependent on preclinical in vivo animal studies. Small animal imaging is invaluable for the identification of new disease markers and the evaluation of drug efficacy. Here, we report for the first time the use of a three-dimensional fluorescence bioimager called FLuorescence Emission Computed Tomography (FLECT) for the detection of a novel recombinant fluoroprobe that is safe, easily prepared on a large scale and stably stored prior to scan. This novel fluoroprobe (Targ-Cy7) comprises a single-chain antibody-fragment (scFvTarg), which binds exclusively to activated-platelets, conjugated to a near-infrared (NIR) dye, Cy7, for detection. Upon mouse carotid artery injury, the injected fluoroprobe circulates and binds within the platelet-rich thrombus. This specific in vivo binding of the fluoroprobe to the thrombus, compared to its non-targeting control-fluoroprobe, is detected by the FLECT imager. The analyzed FLECT image quantifies the NIR signal and localizes it to the site of vascular injury. The detected fluorescence is further verified using a two-dimensional IVIS® Lumina scanner, where significant NIR fluorescence is detected in vivo at the thrombotic site, and ex vivo, at the injured carotid artery. Furthermore, fluorescence levels in various organs have also been quantified for biodistribution, with the highest fluoroprobe uptake shown to be in the injured artery. Subsequently, this live animal imaging technique is successfully employed to monitor the response of the induced thrombus to treatment over time. This demonstrates the potential of using longitudinal FLECT scanning to examine the efficacy of candidate drugs in preclinical settings. Besides intravascular thrombosis, we have shown that this non-invasive FLECT-imaging can also detect in vivo pulmonary embolism. Overall, this report describes a novel fluorescence-based preclinical imaging modality that uses an easy-to-prepare and non-radioactive recombinant fluoroprobe. This represents a unique tool to study mechanisms of thromboembolic diseases and it will strongly facilitate the in vivo testing of antithrombotic drugs. Furthermore, the non-radiation nature, low-cost, high sensitivity, and the rapid advancement of optical scanning technologies make this fluorescence imaging an attractive development for future clinical applications.
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    Activated platelets in the tumor microenvironment for targeting of antibody-drug conjugates to tumors and metastases
    Yap, ML ; McFadyen, JD ; Wang, X ; Ziegler, M ; Chen, Y-C ; Willcox, A ; Nowell, CJ ; Scott, AM ; Sloan, EK ; Hogarth, PM ; Pietersz, GA ; Peter, K (IVYSPRING INT PUBL, 2019)
    Rationale: Platelets are increasingly recognized as mediators of tumor growth and metastasis. Hypothesizing that activated platelets in the tumor microenvironment provide a targeting epitope for tumor-directed chemotherapy, we developed an antibody-drug conjugate (ADC), comprised of a single-chain antibody (scFv) against the platelet integrin GPIIb/IIIa (scFvGPIIb/IIIa) linked to the potent chemotherapeutic microtubule inhibitor, monomethyl auristatin E (MMAE). Methods: We developed an ADC comprised of three components: 1) A scFv which specifically binds to the high affinity, activated integrin GPIIb/IIIa on activated platelets. 2) A highly potent microtubule inhibitor, monomethyl auristatin E. 3) A drug activation/release mechanism using a linker cleavable by cathepsin B, which we demonstrate to be abundant in the tumor microenvironment. The scFvGPIIb/IIIa-MMAE was first conjugated with Cyanine7 for in vivo imaging. The therapeutic efficacy of the scFvGPIIb/IIIa-MMAE was then tested in a mouse metastasis model of triple negative breast cancer. Results: In vitro studies confirmed that this ADC specifically binds to activated GPIIb/IIIa, and cathepsin B-mediated drug release/activation resulted in tumor cytotoxicity. In vivo fluorescence imaging demonstrated that the newly generated ADC localized to primary tumors and metastases in a mouse xenograft model of triple negative breast cancer, a difficult to treat tumor for which a selective tumor-targeting therapy remains to be clinically established. Importantly, we demonstrated that the scFvGPIIb/IIIa-MMAE displays marked efficacy as an anti-cancer agent, reducing tumor growth and preventing metastatic disease, without any discernible toxic effects. Conclusion: Here, we demonstrate the utility of a novel ADC that targets a potent cytotoxic drug to activated platelets and specifically releases the cytotoxic agent within the confines of the tumor. This unique targeting mechanism, specific to the tumor microenvironment, holds promise as a novel therapeutic approach for the treatment of a broad range of primary tumors and metastatic disease, particularly for tumors that lack specific molecular epitopes for drug targeting.
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    Platelet-Derived Microvesicles in Cardiovascular Diseases
    Zaldivia, MTK ; McFadyen, JD ; Lim, B ; Wang, X ; Peter, K (FRONTIERS MEDIA SA, 2017-11-21)
    Microvesicles (MVs) circulating in the blood are small vesicles (100-1,000 nm in diameter) derived from membrane blebs of cells such as activated platelets, endothelial cells, and leukocytes. A growing body of evidence now supports the concept that platelet-derived microvesicles (PMVs), the most abundant MVs in the circulation, are important regulators of hemostasis, inflammation, and angiogenesis. Compared with healthy individuals, a large increase of circulating PMVs has been observed, particularly in patients with cardiovascular diseases. As observed in MVs from other parent cells, PMVs exert their biological effects in multiple ways, such as triggering various intercellular signaling cascades and by participating in transcellular communication by the transfer of their "cargo" of cytoplasmic components and surface receptors to other cell types. This review describes our current understanding of the potential role of PMVs in mediating hemostasis, inflammation, and angiogenesis and their consequences on the pathogenesis of cardiovascular diseases, such as atherosclerosis, myocardial infarction, and venous thrombosis. Furthermore, new developments of the therapeutic potential of PMVs for the treatment of cardiovascular diseases will be discussed.
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    Nanoliposomes for Safe and Efficient Therapeutic mRNA Delivery: A Step Toward Nanotheranostics in Inflammatory and Cardiovascular Diseases as well as Cancer.
    Abraham, M-K ; Peter, K ; Michel, T ; Wendel, HP ; Krajewski, S ; Wang, X (Ivyspring International Publisher, 2017)
    Rationale: Genetic therapy using modified mRNA for specific therapeutic protein expression for disease treatment and vaccination represents a new field of therapeutic and diagnostic medicine. Non-viral vectors transfection using biocompatible nanoliposomes enables safe and efficient delivery of therapeutic mRNA. Objective: Generation of non-toxic, cell-compatible cationic nanoliposomes as nanotheranostic agents to successfully deliver therapeutic mRNA. Methods and results: Cationic nanoliposomes (DC-Cholesterol/DOPE) were generated as transfection vehicles for either eGFP mRNA or the therapeutic anti-inflammatory, CD39 mRNA. We observed no toxicity using these nanoplexes and noted high cell viability after transfection. Nanoplexes for the transfection of eGFP mRNA showed an increase in fluorescence signals on microscopy as compared to the mRNA control after 24 hours in Chinese hamster ovary (CHO) cells (14.29 ± 5.30 vs. 1.49 ± 0.54; mean ± SD respectively; p<0.001) and flow cytometry (57.29 ± 14.59 vs 1.83 ± 0.34; % mean ± SD; p<0.001). Nanoplexes for the transfection of CD39 mRNA showed increased CD39 expression in flow cytometry (45.64 ± 15.3 vs. 3.94 ± 0.45; % mean ± SD; p<0.001) as compared to the mRNA control after 24 hours using CHO cells. We also demonstrated efficient transfection across several cell lines (CHO, HEK293, and A549), as well as long-term protein expression (120 h and 168 h) using these nanoplexes. Conclusions: We have developed and tested non-toxic, safe, and efficient nanoliposome preparations for the delivery of therapeutic mRNA that hold promise for novel therapies in diseases such as inflammatory and cardiovascular diseases, as well as cancer. We have also demonstrated that this approach provides a reliable technology to deliver CD39 mRNA as an anti-inflammatory therapeutic for future nanotheranostics approaches.
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    Mending the failing heart
    Wang, X ; Peter, K (IMPACT JOURNALS LLC, 2019-03-31)
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    Platelets in cardiac ischaemia/reperfusion injury: a promising therapeutic target
    Ziegler, M ; Wang, X ; Peter, K (OXFORD UNIV PRESS, 2019-06-01)
    Acute myocardial infarction (AMI) is the single leading cause of mortality and morbidity worldwide. A key component of AMI therapy is the timely reopening of occluded vessels to prevent further ischaemic damage to the myocardium. However, reperfusion of the ischaemic myocardium can itself trigger reperfusion injury causing up to 50% of the overall infarct size. In recent years, considerable research has been devoted to understanding the pathogenesis of ischaemia/reperfusion (I/R) injury and platelets have emerged as a major contributing factor. This review summarizes the role of platelets in the pathogenesis of I/R injury and highlights the potential of platelet-directed therapeutics to minimize cardiac I/R injury. Activated platelets infiltrate specifically into the ischaemic/reperfused myocardium and contribute to I/R injury by the formation of microthrombi, enhanced platelet-leucocyte aggregation, and the release of potent vasoconstrictor and pro-inflammatory molecules. This review demonstrates the benefits of platelet inhibition beyond their well-described anti-thrombotic effect and highlights the direct cardioprotective role of anti-platelet drugs. In particular, the inhibition of COX, the P2Y12 receptor and the GPIIb/IIIa receptor has demonstrated the potential to attenuate I/R injury. Moreover, targeting of drug candidates or regenerative cells to the activated platelets accumulated within the ischaemic/reperfused myocardium shows remarkable potential to protect the myocardium from I/R injury. Overall, activated platelets play a key role in the pathogenesis of I/R injury. Their direct inhibition as well as their use as epitopes for site-directed therapy is a unique and promising therapeutic approach for the prevention of I/R injury and ultimately the preservation of cardiac function.
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    Cationic Nanoliposomes Meet mRNA: Efficient Delivery of Modified mRNA Using Hemocompatible and Stable Vectors for Therapeutic Applications
    Michel, T ; Luft, D ; Abraham, M-K ; Reinhardt, S ; Medina, MLS ; Kurz, J ; Schaller, M ; Avci-Adali, M ; Schlensak, C ; Peter, K ; Wendel, HP ; Wang, X ; Krajewski, S (CELL PRESS, 2017-09-15)
    Synthetically modified mRNA is a unique bioactive agent, ideal for use in therapeutic applications, such as cancer vaccination or treatment of single-gene disorders. In order to facilitate mRNA transfections for future therapeutic applications, there is a need for the delivery system to achieve optimal transfection efficacy, perform with durable stability, and provide drug safety. The objective of our study was to comprehensively analyze the use of 3β-[N-(N',N'-dimethylaminoethane) carbamoyl](DC-Cholesterol)/dioleoylphosphatidylethanolamine (DOPE) liposomes as a potential transfection agent for modified mRNAs. Our cationic liposomes facilitated a high degree of mRNA encapsulation and successful cell transfection efficiencies. More importantly, no negative effects on cell viability or immune reactions were detected posttransfection. Notably, the liposomes had a long-acting transfection effect on cells, resulting in a prolonged protein production of alpha-1-antitrypsin (AAT). In addition, the stability of these mRNA-loaded liposomes allowed storage for 80 days, without the loss of transfection efficacy. Finally, comprehensive analysis showed that these liposomes are fully hemocompatible with fresh human whole blood. In summary, we present an extensive analysis on the use of DC-cholesterol/DOPE liposomes as mRNA delivery vehicles. This approach provides the basis of a safe and efficient therapeutic strategy in the development of successful mRNA-based drugs.
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    Platelet Depletion is Effective in Ameliorating Anxiety-Like Behavior and Reducing the Pro-Inflammatory Environment in the Hippocampus in Murine Experimental Autoimmune Encephalomyelitis
    Kocovski, P ; Jiang, X ; D'Souza, CS ; Li, Z ; Dang, PT ; Wang, X ; Chen, W ; Peter, K ; Hale, MW ; Orian, JM (MDPI, 2019-02)
    The neuropsychiatric symptoms of multiple sclerosis (MS), such as anxiety and depression, can result from disease activity itself as well as psychological reaction to an unfavorable diagnosis. Accordingly, the literature reports evidence of increased anxiety-like behavior in experimental autoimmune encephalomyelitis (EAE), an accepted MS model. Due to the recently described critical role of platelets in inflammation and autoimmune disease, we examined the relationship between platelets, inflammation, and anxiety-like behavior in EAE. In the elevated plus maze, EAE-induced C57BL/6J mice showed decreased time spent in the open arms relative to vehicle-only controls, demonstrating an increase in anxiety-like behavior. This effect occurred in the presence of platelet⁻neuron association, but absence of lymphocytic infiltration, in the hippocampal parenchyma. Platelet depletion at the pre-clinical disease stage, using antibody-mediated lysis prevented the EAE-induced increase in anxiety-like behavior, while no significant difference in distance moved was recorded. Furthermore, platelet depletion was also associated with reduction of the pro-inflammatory environment to control levels in the hippocampus and prevention of EAE disease symptomology. These studies demonstrate the high efficacy of a platelet-targeting approach in preventing anxiety-like symptoms and clinical manifestations of EAE and have implications for the treatment of neuropsychiatric symptoms in MS.