scholarly journals Ultrasound Molecular Imaging of Atherosclerosis With Nanobodies

2019 ◽  
Vol 39 (12) ◽  
pp. 2520-2530 ◽  
Author(s):  
Mukesh Punjabi ◽  
Lifen Xu ◽  
Amanda Ochoa-Espinosa ◽  
Alexandra Kosareva ◽  
Thomas Wolff ◽  
...  
Keyword(s):  
Shear Stress ◽  
Molecular Imaging ◽  
Pulsatile Flow ◽  
Ex Vivo ◽  
Circulation Model ◽  
Wild Type ◽  
Double Knockout ◽  
Ultrasound Molecular Imaging

Objective: Contrast-enhanced ultrasound molecular imaging (CEUMI) of endothelial expression of VCAM (vascular cell adhesion molecule)-1 could improve risk stratification for atherosclerosis. The microbubble contrast agents developed for preclinical studies are not suitable for clinical translation. Our aim was to characterize and validate a microbubble contrast agent using a clinically translatable single-variable domain immunoglobulin (nanobody) ligand. Approach and Results: Microbubble with a nanobody targeting VCAM-1 (MB cAbVcam1-5 ) and microbubble with a control nanobody (MB VHH2E7 ) were prepared and characterized in vitro. Attachment efficiency to VCAM-1 under continuous and pulsatile flow was investigated using activated murine endothelial cells. In vivo CEUMI of the aorta was performed in atherosclerotic double knockout and wild-type mice after injection of MB cAbVcam1-5 and MB VHH2E7 . Ex vivo CEUMI of human endarterectomy specimens was performed in a closed-loop circulation model. The surface density of the nanobody ligand was 3.5×10 5 per microbubble. Compared with MB VHH2E7 , MB cAbVcam1-5 showed increased attachment under continuous flow with increasing shear stress of 1-8 dynes/cm 2 while under pulsatile flow attachment occurred at higher shear stress. CEUMI in double knockout mice showed signal enhancement for MB cAbVcam1-5 in early ( P =0.0003 versus MB VHH2E7 ) and late atherosclerosis ( P =0.007 versus MB VHH2E7 ); in wild-type mice, there were no differences between MB cAbVcam1-5 and MB VHH2E7 . CEUMI in human endarterectomy specimens showed a 100% increase in signal for MB cAbVcam1-5 versus MB VHH2E7 (20.6±27.7 versus 9.6±14.7, P =0.0156). Conclusions: CEUMI of the expression of VCAM-1 is feasible in murine models of atherosclerosis and on human tissue using a clinically translatable microbubble bearing a VCAM-1 targeted nanobody.

Ultrasound molecular imaging of arterial thrombi with novel microbubbles modified by cyclic RGD in vitro and in vivo

2012 ◽  
Vol 107 (01) ◽  
pp. 172-183 ◽  
Author(s):  
Yulin Liao ◽  
Li Yang ◽  
Ruizhu Huang ◽  
Juefei Wu ◽  
Jiajia Xie ◽  
...  
Keyword(s):  
Shear Stress ◽  
Molecular Imaging ◽  
Molecular Probe ◽  
Great Promise ◽  
Rgd Peptides ◽  
Abdominal Aortic ◽  
Aortic Thrombus ◽  
Ultrasound Molecular Imaging

SummaryDespite immense potential, ultrasound molecular imaging (UMI) of arterial thrombi remains very challenging because the high-shear arterial flow limits binding of site-targeted microbubbles to the thrombi. The linear Arg-Gly-Asp (RGD) peptides have been successfully applied to evaluate venous, atrial, and arteriolar thrombi, but have thus far failed in the detection of arterial thrombi. Cyclic RGD (Arg-Gly-Asp-D-Phe-Cys) is a cyclic conformation of linear RGD peptides, which has much higher binding-affinity and selectivity for binding to the glycoprotein (GP) IIb/IIIa receptor than its linear counterpart and thus is likely to be an optimal targeted molecular probe for ultrasound molecular imaging of arterial thrombi. In this study, we sought to assess the feasibility of a novel microbubble conjugated with cyclic RGD (Mb-cyclic RGD) in UMI of arterial thrombi in vitro and in vivo. As expected, Mb-cyclic RGD had greater GP IIb/IIIa-targeted binding capability in all shear stress conditions. In addition, the shear stress at half-maximal detachment of Mb-cyclic RGD was 5.7-fold higher than that of microbubbles with nonspecific peptide (Mb-CON) (p<0.05). Mb-cyclic RGD enhanced the echogenicity of the platelet-rich thrombus in vitro whereas Mb-CON did not produce enhancement. In the in vivo setting, optimal signal enhancement of the abdominal aortic thrombus was displayed with Mb-cyclic RGD in all cases. Mean video intensity of the abdominal aortic thrombi with Mb-cyclic RGD was 3.2-fold higher than that with Mb-CON (p<0.05). The novel Mb-cyclic RGD facilitated excellent visualisation of arterial thrombi using UMI and showed great promise for clinical applications.

scholarly journals Differential effect of anesthetics on mucociliary clearance in vivo in mice

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Kyle S. Feldman ◽  
Eunwon Kim ◽  
Michael J. Czachowski ◽  
Yijen Wu ◽  
Cecilia W. Lo ◽  
...  
Keyword(s):  
Mucociliary Clearance ◽  
Defense Mechanism ◽  
Ex Vivo ◽  
Beat Frequency ◽  
Ciliary Beat Frequency ◽  
Wild Type ◽  
Sulfur Colloid ◽  
Ct System

AbstractRespiratory mucociliary clearance (MCC) is a key defense mechanism that functions to entrap and transport inhaled pollutants, particulates, and pathogens away from the lungs. Previous work has identified a number of anesthetics to have cilia depressive effects in vitro. Wild-type C57BL/6 J mice received intra-tracheal installation of 99mTc-Sulfur colloid, and were imaged using a dual-modality SPECT/CT system at 0 and 6 h to measure baseline MCC (n = 8). Mice were challenged for one hour with inhalational 1.5% isoflurane, or intraperitoneal ketamine (100 mg/kg)/xylazine (20 mg/kg), ketamine (0.5 mg/kg)/dexmedetomidine (50 mg/kg), fentanyl (0.2 mg/kg)/1.5% isoflurane, propofol (120 mg/Kg), or fentanyl/midazolam/dexmedetomidine (0.025 mg/kg/2.5 mg/kg/0.25 mg/kg) prior to MCC assessment. The baseline MCC was 6.4%, and was significantly reduced to 3.7% (p = 0.04) and 3.0% (p = 0.01) by ketamine/xylazine and ketamine/dexmedetomidine challenge respectively. Importantly, combinations of drugs containing fentanyl, and propofol in isolation did not significantly depress MCC. Although no change in cilia length or percent ciliation was expected, we tried to correlate ex-vivo tracheal cilia ciliary beat frequency and cilia-generated flow velocities with MCC and found no correlation. Our results indicate that anesthetics containing ketamine (ketamine/xylazine and ketamine/dexmedetomidine) significantly depress MCC, while combinations containing fentanyl (fentanyl/isoflurane, fentanyl/midazolam/dexmedetomidine) and propofol do not. Our method for assessing MCC is reproducible and has utility for studying the effects of other drug combinations.

Abstract P283: Parkin Mediates Mitophagy in Cardioprotection

2011 ◽  
Vol 109 (suppl_1) ◽  
Author(s):  
Allen M Andres ◽  
Chengqun Huang ◽  
Eric P Ratliff ◽  
Genaro Hernandez ◽  
Pamela Lee ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Wild Type ◽  
Simulated Ischemia ◽  
Selective Targeting ◽  
Cardioprotective Effects ◽  
Mitochondrial Turnover ◽  
First Time

Autophagy-dependent mitochondrial turnover in response to cellular stress is necessary for maintaining cellular homeostasis. However, the mechanisms that govern the selective targeting of damaged mitochondria are poorly understood. Parkin, an E3 ubiquitin ligase, has been shown to be essential for the selective clearance of damaged mitochondria. Parkin is expressed in the heart, yet its function has not been investigated in the context of cardioprotection. We previously reported that autophagy is required for cardioprotection by ischemic preconditioning (IPC). In the present study, we used simulated ischemia in vitro and IPC in hearts (in vivo and ex vivo) to investigate the role of Parkin in mediating cardioprotection. In HL-1 cells, simulated ischemia induced Parkin translocation to mitochondria and mitochondrial elimination. Mitochondrial loss was blunted in Atg5-deficient cells, revealing the requirement for autophagy in mitochondrial elimination. Consistent with previous reports implicating p62/SQSTM1 in mitophagy, we found that downregulation of p62 attenuated mitophagy and exacerbated cell death in HL-1 cardiomyocytes subjected to simulated ischemia. While wild type mice showed p62 translocation to mitochondria after IPC, Parkin knockout mice exhibited attenuated translocation of p62 to mitochondria. Importantly, ablation of Parkin in mice abolished the cardioprotective effects of IPC. These results reveal for the first time the crucial role of Parkin and mitophagy in cardioprotection.

scholarly journals Proof of concept of a multimodal intravital molecular imaging system for tumour transpathology investigation

2021 ◽  
Author(s):  
Zhen Liu ◽  
Tao Cheng ◽  
Stephan Düwel ◽  
Ziying Jian ◽  
Geoffrey J. Topping ◽  
...  
Keyword(s):  
Molecular Imaging ◽  
Imaging System ◽  
Ex Vivo ◽  
Fiducial Markers ◽  
Proof Of Concept ◽  
Registration Accuracy ◽  
Microscopic Imaging ◽  
Window Chamber

Abstract Background Transpathology highlights the interpretation of the underlying physiology behind molecular imaging. However, it remains challenging due to the discrepancies between in vivo and in vitro measurements and difficulties of precise co-registration between trans-scaled images. This study aims to develop a multimodal intravital molecular imaging (MIMI) system as a tool for in vivo tumour transpathology investigation. Methods The proposed MIMI system integrates high-resolution positron imaging, magnetic resonance imaging (MRI) and microscopic imaging on a dorsal skin window chamber on an athymic nude rat. The window chamber frame was designed to be compatible with multimodal imaging and its fiducial markers were customized for precise physical alignment among modalities. The co-registration accuracy was evaluated based on phantoms with thin catheters. For proof of concept, tumour models of the human colorectal adenocarcinoma cell line HT-29 were imaged. The tissue within the window chamber was sectioned, fixed and haematoxylin–eosin (HE) stained for comparison with multimodal in vivo imaging. Results The final MIMI system had a maximum field of view (FOV) of 18 mm × 18 mm. Using the fiducial markers and the tubing phantom, the co-registration errors are 0.18 ± 0.27 mm between MRI and positron imaging, 0.19 ± 0.22 mm between positron imaging and microscopic imaging and 0.15 ± 0.27 mm between MRI and microscopic imaging. A pilot test demonstrated that the MIMI system provides an integrative visualization of the tumour anatomy, vasculatures and metabolism of the in vivo tumour microenvironment, which was consistent with ex vivo pathology. Conclusions The established multimodal intravital imaging system provided a co-registered in vivo platform for trans-scale and transparent investigation of the underlying pathology behind imaging, which has the potential to enhance the translation of molecular imaging.

scholarly journals An Ex Vivo Vessel Injury Model to Study Remodeling

2018 ◽  
Vol 27 (9) ◽  
pp. 1375-1389 ◽  
Author(s):  
Mehmet H. Kural ◽  
Guohao Dai ◽  
Laura E. Niklason ◽  
Liqiong Gui
Keyword(s):  
Shear Stress ◽  
Animal Models ◽  
Intimal Hyperplasia ◽  
Vascular Remodeling ◽  
Ex Vivo ◽  
Vessel Injury ◽  
Injury Model ◽  
Human Arteries

Objective: Invasive coronary interventions can fail due to intimal hyperplasia and restenosis. Endothelial cell (EC) seeding to the vessel lumen, accelerating re-endothelialization, or local release of mTOR pathway inhibitors have helped reduce intimal hyperplasia after vessel injury. While animal models are powerful tools, they are complex and expensive, and not always reflective of human physiology. Therefore, we developed an in vitro 3D vascular model validating previous in vivo animal models and utilizing isolated human arteries to study vascular remodeling after injury. Approach: We utilized a bioreactor that enables the control of intramural pressure and shear stress in vessel conduits to investigate the vascular response in both rat and human arteries to intraluminal injury. Results: Culturing rat aorta segments in vitro, we show that vigorous removal of luminal ECs results in vessel injury, causing medial proliferation by Day-4 and neointima formation, with the observation of SCA1+ cells (stem cell antigen-1) in the intima by Day-7, in the absence of flow. Conversely, when endothelial-denuded rat aortae and human umbilical arteries were subjected to arterial shear stress, pre-seeding with human umbilical ECs decreased the number and proliferation of smooth muscle cell (SMC) significantly in the media of both rat and human vessels. Conclusion: Our bioreactor system provides a novel platform for correlating ex vivo findings with vascular outcomes in vivo. The present in vitro human arterial injury model can be helpful in the study of EC-SMC interactions and vascular remodeling, by allowing for the separation of mechanical, cellular, and soluble factors.

An Innovative Ex Vivo Vascular Bioreactor as Comprehensive Tool to Study the Behavior of Native Blood Vessels Under Physiologically Relevant Conditions

2019 ◽  
Vol 2 (4) ◽  
Author(s):  
Noemi Vanerio ◽  
Marco Stijnen ◽  
Bas A. J. M. de Mol ◽  
Linda M. Kock
Keyword(s):  
Shear Stress ◽  
Blood Vessels ◽  
Ultrasound Imaging ◽  
Ex Vivo ◽  
Biological Response ◽  
Vessel Diameter ◽  
Tissue Growth ◽  
In Vivo Models

Abstract Ex vivo systems represent important models to study vascular biology and to test medical devices, combining the advantages of in vitro and in vivo models such as controllability of parameters and the presence of biological response, respectively. The aim of this study was to develop a comprehensive ex vivo vascular bioreactor to long-term culture and study the behavior of native blood vessels under physiologically relevant conditions. The system was designed to allow for physiological mechanical loading in terms of pulsatile hemodynamics, shear stress, and longitudinal prestretch and ultrasound imaging for vessel diameter and morphology evaluation. In this first experience, porcine carotid arteries (n = 4) from slaughterhouse animals were cultured in the platform for 10 days at physiological temperature, CO2 and humidity using medium with blood-mimicking viscosity, components, and stability of composition. As expected, a significant increase in vessel diameter was observed during culture. Flow rate was adjusted according to diameter values to reproduce and maintain physiological shear stress, while pressure was kept physiological. Ultrasound imaging showed that the morphology and structure of cultured arteries were comparable to in vivo. Histological analyses showed preserved endothelium and extracellular matrix and neointimal tissue growth over 10 days of culture. In conclusion, we have developed a comprehensive pulsatile system in which a native blood vessel can be cultured under physiological conditions. The present model represents a significant step toward ex vivo testing of vascular therapies, devices, drug interaction, and as basis for further model developments.

Development of Megakaryocyte-Specific Lentiviral Vectors for Gene Therapy of Platelet Disorders.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 5143-5143
Author(s):  
Liesbeth De Waele ◽  
Kathleen Freson ◽  
Chantal Thys ◽  
Christel Van Geet ◽  
Désiré Collen ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Transgene Expression ◽  
Ex Vivo ◽  
Phosphoglycerate Kinase ◽  
Lentiviral Vectors ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Platelet Disorders

Abstract The prevalence of congenital platelet disorders has not been established but for some life-threatening bleeding disorders the current therapies are not adequate, justifying the development of alternative strategies as gene therapy. In the case of platelet dysfunction and thrombocytopenia as described for GATA1 deficiency, potentially lethal internal bleedings can occur. The objective of the study is to develop improved lentiviral vectors for megakaryocyte(MK)-specific long term gene expression by ex vivo transduction of hematopoietic stem cells (HSC) to ultimately use for congenital thrombopathies as GATA1 deficiency. Self-inactivating lentiviral vectors were constructed expressing GFP driven by the murine (m) or human (h) GPIIb promoter. These promoters contain multiple Ets and GATA binding sites directing MK-specificity. To evaluate the cell lineage-specificity and transgene expression potential of the vectors, murine Sca1+ and human CD34+ HSC were transduced in vitro with Lenti-hGPIIb-GFP and Lenti-mGPIIb-GFP vectors. After transduction the HSC were induced to differentiate in vitro along the MK and non-MK lineages. The mGPIIb and hGPIIb promoters drove GFP expression at overall higher levels (20% in murine cells and 25% in human cells) than the ubiquitous CMV (cytomegalovirus) or PGK (phosphoglycerate kinase) promoters, and this exclusively in the MK lineage. Interestingly, in both human and murine HSC the hGPIIb promoter with an extra RUNX and GATA binding site, was more potent in the MK lineage compared to the mGPIIb promoter. Since FLI1 and GATA1 are the main transcription factors regulating GPIIb expression, we tested the Lenti-hGPIIb-GFP construct in GATA1 deficient HSC and obtained comparable transduction efficiencies as for wild-type HSC. To assess the MK-specificity of the lentiviral vectors in vivo, we transplanted irradiated wild-type C57Bl/6 mice with Sca1+ HSC transduced with the Lenti-hGPIIb-GFP constructs. Six months after transplantation we could detect 6% GFP positive platelets without a GFP signal in other cell lineages. Conclusion: In vitro and in vivo MK-specific transgene expression driven by the hGPIIb and mGPIIb promoters could be obtained after ex vivo genetic engineering of HSC by improved lentiviral vectors. Studies are ongoing to study whether this approach can induce phenotypic correction of GATA1 deficient mice by transplantation of ex vivo Lenti-hGPIIb-GATA1 transduced HSC.

scholarly journals Thyroid Cancer Detection by Ultrasound Molecular Imaging with SHP2-Targeted Perfluorocarbon Nanoparticles

2018 ◽  
Vol 2018 ◽  
pp. 1-7 ◽  
Author(s):  
ZhongQian Hu ◽  
Bin Yang ◽  
Tiankuan Li ◽  
Jia Li
Keyword(s):  
Thyroid Cancer ◽  
Molecular Imaging ◽  
Diagnostic Accuracy ◽  
Protein Tyrosine Phosphatases ◽  
Ultrasound Contrast Agents ◽  
Human Thyroid ◽  
Thyroid Tumour ◽  
Ultrasound Molecular Imaging

Background. Contrast-enhanced ultrasound imaging has been widely used in the ultrasound diagnosis of a variety of tumours with high diagnostic accuracy, especially in patients with hepatic carcinoma, while its application is rarely reported in thyroid cancer. The currently used ultrasound contrast agents, microbubbles, cannot be targeted to molecular markers expressed in tumour cells due to their big size, leading to a big challenge for ultrasound molecular imaging. Phase-changeable perfluorocarbon nanoparticles may resolve the penetrability limitation of microbubbles and serve as a promising probe for ultrasound molecular imaging. Methods. 65 thyroid tumour samples and 40 normal samples adjacent to thyroid cancers were determined for SHP2 expression by IHC. SHP2-targeted PLGA nanoparticles (NPs-SHP2) encapsulating perfluoropentane (PFP) were prepared with PLGA-PEG as a shell material, and their specific target-binding ability was assessed in vitro and in vivo, and the effect on the enhancement of ultrasonic imaging induced by LIFU was studied in vivo. Results. In the present study, we verified that tumour overexpression of SHP2 and other protein tyrosine phosphatases regulated several cellular processes and contributed to tumorigenesis, which could be introduced to ultrasound molecular imaging for differentiating normal from malignant thyroid diagnostic nodes. The IHC test showed remarkably high expression of SHP2 in human thyroid carcinoma specimens. In thyroid tumour xenografts in mice, the imaging signal was significantly enhanced by SHP2-targeted nanoparticles after LIFU induction. Conclusion. This study provides a basis for preclinical exploration of ultrasound molecular imaging with NPs-SHP2 for clinical thyroid nodule detection to enhance diagnostic accuracy.

Autonomous Effects of Shear Stress and Cyclic Circumferential Stretch Regarding Endothelial Dysfunction and Oxidative Stress: An Ex Vivo Arterial Model

2009 ◽  
Author(s):  
Tyler Thacher ◽  
Rafaela da Silva ◽  
Paolo Silacci ◽  
Nikos Stergiopulos
Keyword(s):  
Shear Stress ◽  
Endothelial Dysfunction ◽  
Ex Vivo ◽  
Cyclic Stretch ◽  
Arterial Model ◽  
Individual Contributions ◽  
Independent Effects ◽  
And Oxidative Stress

Within the vasculature endothelial cells are constantly exposed to dynamic mechanical forces generated by pulsatile blood flow. Two stimuli known to modulate endothelial function are shear stress and cyclic circumferential strain. Yet, in most studies these two stimuli are simultaneously coupled in-vivo, making it very difficult to understand their individual contributions to vascular disease. Some attempts have been made to de-couple stretch and shear stress in-vitro by using different cell lines in a variety of stretch systems and flow chambers, straying from reality and making it hard to draw definitive conclusions. In this study we wish to find a compromise between the in-vivo and in-vitro work of the past by studying the independent effects of shear stress and cyclic stretch and how they contribute to endothelial dysfunction.

scholarly journals TLR9 regulates Th1 responses and cooperates with TLR2 in mediating optimal resistance to Mycobacterium tuberculosis

2005 ◽  
Vol 202 (12) ◽  
pp. 1715-1724 ◽  
Author(s):  
Andre Bafica ◽  
Charles A. Scanga ◽  
Carl G. Feng ◽  
Cynthia Leifer ◽  
Allen Cheever ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Ex Vivo ◽  
Double Knockout ◽  
Susceptibility To Infection ◽  
Pathogen Challenge ◽  
In Vitro Response ◽  
Ifn Γ ◽  
Aerosol Infection

To investigate the role of Toll-like receptor (TLR)9 in the immune response to mycobacteria as well as its cooperation with TLR2, a receptor known to be triggered by several major mycobacterial ligands, we analyzed the resistance of TLR9−/− as well as TLR2/9 double knockout mice to aerosol infection with Mycobacterium tuberculosis. Infected TLR9−/− but not TLR2−/− mice displayed defective mycobacteria-induced interleukin (IL)-12p40 and interferon (IFN)-γ responses in vivo, but in common with TLR2−/− animals, the TLR9−/− mice exhibited only minor reductions in acute resistance to low dose pathogen challenge. When compared with either of the single TLR-deficient animals, TLR2/9−/− mice displayed markedly enhanced susceptibility to infection in association with combined defects in proinflammatory cytokine production in vitro, IFN-γ recall responses ex vivo, and altered pulmonary pathology. Cooperation between TLR9 and TLR2 was also evident at the level of the in vitro response to live M. tuberculosis, where dendritic cells and macrophages from TLR2/9−/− mice exhibited a greater defect in IL-12 response than the equivalent cell populations from single TLR9-deficient animals. These findings reveal a previously unappreciated role for TLR9 in the host response to M. tuberculosis and illustrate TLR collaboration in host resistance to a major human pathogen.

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