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Bart Spronck, Abhay B. Ramachandra, Lauren Moriyama, Jakub Toczek, Jinah Han, Mehran M. Sadeghi, and Jay D. Humphrey
Journal of Biomechanics, ISSN: 00219290, eISSN: 18732380, Volume: 141, Published: August 2022 Elsevier BV
Xinbo Zhang, Noemi Rotllan, Alberto Canfrán-Duque, Jonathan Sun, Jakub Toczek, Anna Moshnikova, Shipra Malik, Nathan L. Price, Elisa Araldi, Wen Zhong, Mehran M. Sadeghi, Oleg A. Andreev, Raman Bahal, Yana K. Reshetnyak, Yajaira Suárez, and Carlos Fernández-Hernando
Circulation Research, ISSN: 00097330, eISSN: 15244571, Volume: 131, Pages: 77-90, Published: 24 June 2022 Ovid Technologies (Wolters Kluwer Health)
Background: miRNA therapeutics have gained attention during the past decade. These oligonucleotide treatments can modulate the expression of miRNAs in vivo and could be used to correct the imbalance of gene expression found in human diseases such as obesity, metabolic syndrome, and atherosclerosis. The in vivo efficacy of current anti-miRNA technologies hindered by physiological and cellular barriers to delivery into targeted cells and the nature of miRNAs that allows one to target an entire pathway that may lead to deleterious off-target effects. For these reasons, novel targeted delivery systems to inhibit miRNAs in specific tissues will be important for developing effective therapeutic strategies for numerous diseases including atherosclerosis. Methods: We used pH low-insertion peptide (pHLIP) constructs as vehicles to deliver microRNA-33-5p (miR-33) antisense oligonucleotides to atherosclerotic plaques. Immunohistochemistry and histology analysis was performed to assess the efficacy of miR-33 silencing in atherosclerotic lesions. We also assessed how miR-33 inhibition affects gene expression in monocytes/macrophages by single-cell RNA transcriptomics. Results: The anti-miR-33 conjugated pHLIP constructs are preferentially delivered to atherosclerotic plaque macrophages. The inhibition of miR-33 using pHLIP-directed macrophage targeting improves atherosclerosis regression by increasing collagen content and decreased lipid accumulation within vascular lesions. Single-cell RNA sequencing analysis revealed higher expression of fibrotic genes ( Col2a1, Col3a1, Col1a2, Fn1 , etc) and tissue inhibitor of metalloproteinase 3 ( Timp3 ) and downregulation of Mmp12 in macrophages from atherosclerotic lesions targeted by pHLIP-anti-miR-33. Conclusions: This study provides proof of principle for the application of pHLIP for treating advanced atherosclerosis via pharmacological inhibition of miR-33 in macrophages that avoid the deleterious effects in other metabolic tissues. This may open new therapeutic opportunities for atherosclerosis-associated cardiovascular diseases via selective delivery of other protective miRNAs.
Jae-Joon Jung, Azmi A. Ahmad, Saranya Rajendran, Linyan Wei, Jiasheng Zhang, Jakub Toczek, Lei Nie, Gunjan Kukreja, Mani Salarian, Kiran Gona, Mean Ghim, Raja Chakraborty, Kathleen A. Martin, George Tellides, Donald Heistad, and Mehran M. Sadeghi
JACC: Basic to Translational Science, ISSN: 2452302X, Pages: 333-345, Published: April 2022 Elsevier BV
Journal of Nuclear Cardiology, ISSN: 10713581, eISSN: 15326551, Pages: 1946-1948, Published: October 2021 Springer Science and Business Media LLC
Jakub Toczek, Ansel T. Hillmer, Jinah Han, Chi Liu, Dana Peters, Hamed Emami, Jing Wu, Irina Esterlis, Kelly P. Cosgrove, and Mehran M. Sadeghi
Journal of Nuclear Cardiology, ISSN: 10713581, eISSN: 15326551, Pages: 688-694, Published: April 2021 Springer Science and Business Media LLC
The prevalence of cardiovascular diseases (CVD) is increased in subjects with post-traumatic stress disorder (PTSD). Vascular inflammation mediates CVD and may be assessed by 18F-fluorodeoxyglucose (FDG) positron emission tomography (PET) imaging. In this pilot study, we investigated whether subjects with PTSD have enhanced vascular and systemic inflammation compared to healthy controls, as assessed by FDG PET imaging.MethodsA prospective group of 16 subjects (9 PTSD and 7 controls, age 34 ± 7) without prior history of CVD underwent FDG PET/CT imaging. The presence of PTSD symptoms at the time of the study was confirmed using PTSD checklist for DSM-5 (PCL5) questionnaire. Blood samples were collected to determine blood glucose, lipid and inflammatory biomarkers (tumor necrosis factor α, interleukin-1β, and interleukin-6) levels. FDG signal in the ascending aorta, amygdala, spleen and bone marrow was quantified.ResultsThe two groups matched closely with regards to cardiovascular risk factors. The inflammatory biomarkers were all within the normal range. There was no significant difference in FDG signal in the aorta (target to background ratio: 2.40 ± 0.29 and 2.34 ± 0.29 for control and PTSD subjects, difference: − 0.06, 95% confidence interval of difference: − 0.38 to 0.26), spleen, bone marrow, or amygdala between control and PTSD subjects. There was no significant correlation between aortic and amygdala FDG signal. However, a significant positive correlation existed between amygdala, splenic, and bone marrow FDG signal.ConclusionThis pilot, small study did not reveal any difference in vascular or systemic inflammation as assessed by FDG PET imaging between PTSD and healthy control subjects. Because of the small number of subjects, a modest increase in vascular inflammation, which requires larger scale studies to establish, cannot be excluded. The correlation between FDG signal in amygdala, spleen and bone marrow may reflect a link between amygdala activity and systemic inflammation.
Jakub Toczek, Parnaz Boodagh, Nowshin Sanzida, Mean Ghim, Mani Salarian, Kiran Gona, Gunjan Kukreja, Saranya Rajendran, Linyan Wei, Jinah Han, Jiasheng Zhang, Jae-Joon Jung, Morven Graham, Xinran Liu, and Mehran M. Sadeghi
Theranostics, eISSN: 18387640, Pages: 5876-5888, Published: 2021 Ivyspring International Publisher
Inflammation plays a major role in the pathogenesis of several vascular pathologies, including abdominal aortic aneurysm (AAA). Evaluating the role of inflammation in AAA pathobiology and potentially outcome in vivo requires non-invasive tools for high-resolution imaging. We investigated the feasibility of X-ray computed tomography (CT) imaging of phagocytic activity using nanoparticle contrast agents to predict AAA outcome. Methods: Uptake of several nanoparticle CT contrast agents was evaluated in a macrophage cell line. The most promising agent, Exitron nano 12000, was further characterized in vitro and used for subsequent in vivo testing. AAA was induced in Apoe-/- mice through angiotensin II (Ang II) infusion for up to 4 weeks. Nanoparticle biodistribution and uptake in AAA were evaluated by CT imaging in Ang II-infused Apoe-/- mice. After imaging, the aortic tissue was harvested and used from morphometry, transmission electron microscopy and gene expression analysis. A group of Ang II-infused Apoe-/- mice underwent nanoparticle-enhanced CT imaging within the first week of Ang II infusion, and their survival and aortic external diameter were evaluated at 4 weeks to address the value of vessel wall CT enhancement in predicting AAA outcome. Results: Exitron nano 12000 showed specific uptake in macrophages in vitro. Nanoparticle accumulation was observed by CT imaging in tissues rich in mononuclear phagocytes. Aortic wall enhancement was detectable on delayed CT images following nanoparticle administration and correlated with vessel wall CD68 expression. Transmission electron microscopy ascertained the presence of nanoparticles in AAA adventitial macrophages. Nanoparticle-induced CT enhancement on images obtained within one week of AAA induction was predictive of AAA outcome at 4 weeks. Conclusions: By establishing the feasibility of CT-based molecular imaging of phagocytic activity in AAA, this study links the inflammatory signal on early time point images to AAA evolution. This readily available technology overcomes an important barrier to cross-sectional, longitudinal and outcome studies, not only in AAA, but also in other cardiovascular pathologies and facilitates the evaluation of modulatory interventions, and ultimately upon clinical translation, patient management.
Kiran Gona, Jakub Toczek, Yunpeng Ye, Nowshin Sanzida, Arvene Golbazi, Parnaz Boodagh, Mani Salarian, Jae-Joon Jung, Saranya Rajendran, Gunjan Kukreja, Terence L. Wu, Laurent Devel, and Mehran M. Sadeghi
Journal of Medicinal Chemistry, ISSN: 00222623, eISSN: 15204804, Pages: 15037-15049, Published: 10 December 2020 American Chemical Society (ACS)
Macrophage elastase [matrix metalloproteinase (MMP)-12] is the most upregulated MMP in abdominal aortic aneurysm (AAA) and, hence, MMP-12-targeted imaging may predict AAA progression and rupture risk. Here, we report the design, synthesis, and evaluation of three novel hydroxamate-based selective MMP-12 inhibitors (CGA, CGA-1, and AGA) and the methodology to obtain MMP-12 selectivity from hydroxamate-based panMMP inhibitors. Also, we report two 99mTc-radiotracers, 99mTc-AGA-1 and 99mTc-AGA-2, derived from AGA. 99mTc-AGA-2 displayed faster blood clearance in mice and better radiochemical stability compared to 99mTc-AGA-1. Based on this, 99mTc-AGA-2 was chosen as the lead tracer and tested in murine AAA. 99mTc-AGA-2 uptake detected by autoradiography was significantly higher in AAA compared to normal aortic regions. Specific binding of the tracer to MMP-12 was demonstrated through ex vivo competition. Accordingly, this study introduces a novel family of selective MMP-12 inhibitors and tracers, paving the way for further development of these agents as therapeutic and imaging agents.
Jakub Toczek, Jing Wu, Ansel T. Hillmer, Jinah Han, Irina Esterlis, Kelly P. Cosgrove, Chi Liu, and Mehran M. Sadeghi
Journal of Nuclear Cardiology, ISSN: 10713581, eISSN: 15326551, Pages: 1578-1581, Published: 1 October 2020 Springer Science and Business Media LLC
2-deoxy-2- [18F] fluoro-d-glucose (FDG) PET is commonly used for the assessment of vessel wall inflammation. Guidelines for analysis of arterial wall FDG signal recommend the use of the average of maximal standardized uptake value (mean SUVmax) and target-to-blood (mean TBRmax) ratio. However, these methods have not been validated against a gold standard such as tissue activity ex vivo or net uptake rate of FDG (Ki) obtained using kinetic modeling. We sought to evaluate the accuracy of mean SUVmax and mean TBRmax for aortic wall FDG signal quantification in comparison with the net uptake rate of FDG. Dynamic PET data from 13 subjects without prior history of cardiovascular disease who enrolled in a study of vascular inflammation were used for this analysis. Ex vivo measurement of plasma activity was used as the input function and voxel-by-voxel Patlak analysis was performed with t* = 20 minute to obtain the Ki image. The FDG signal in the ascending aortic wall was quantified on PET images following recent guidelines for vascular imaging to determine mean SUVmax and mean TBRmax. The Ki in the ascending aortic wall did not correlate with mean SUVmax (r = 0.10, P = NS), but correlated with mean TBRmax (r = 0.82, P < 0.001) (Figure 1B). Ki and Ki_max strongly correlated (R = 0.96, P < 0.0001) and similar to Ki, Ki_max did not correlate with mean SUVmax (r = 0.17, P = NS), but correlated with mean TBRmax (r = 0.83, P < 0.001). Kinetic modeling supports the use of mean TBRmax as a surrogate for the net uptake rate of FDG in the arterial wall. These results are relevant to any PET imaging agent, regardless of the biological significance of the tracer uptake in the vessel wall.
Jakub Toczek, Thomas Bordenave, Kiran Gona, Hye-Yeong Kim, Fabrice Beau, Dimitris Georgiadis, Isabelle Correia, Yunpeng Ye, Mahmoud Razavian, Jae-Joon Jung, Olivier Lequin, Vincent Dive, Mehran M. Sadeghi, and Laurent Devel
Journal of Medicinal Chemistry, ISSN: 00222623, eISSN: 15204804, Pages: 9743-9752, Published: 14 November 2019 American Chemical Society (ACS)
Matrix metalloproteinase-12 (MMP-12) is highly upregulated in several inflammatory diseases, including abdominal aortic aneurysm (AAA). Here we report four novel 99mTc-labeled radiotracers derived from a highly selective competitive MMP-12 inhibitor. These tracers in their 99gTc version were assessed in vitro on a set of human metalloproteases and displayed high affinity and selectivity toward MMP-12. Their radiolabeling with 99mTc was shown to be efficient and stable in both buffer and mouse blood. The tracers showed major differences in their biodistribution and blood clearance. Based on its in vivo performance, [99mTc]-1 was selected for evaluation in murine AAA, where MMP-12 gene expression is upregulated. Autoradiography of aortae at two hours post-injection revealed high uptake of [99mTc]-1 in AAA relative to adjacent aorta. Tracer uptake specificity was demonstrated through in vivo competition. This study paves the way for further evaluation of [99mTc]-1 for imaging AAA and other MMP-12-associated diseases.
Nathan L. Price, Verónica Miguel, Wen Ding, Abhishek K. Singh, Shipra Malik, Noemi Rotllan, Anna Moshnikova, Jakub Toczek, Caroline Zeiss, Mehran M. Sadeghi, Noemi Arias, Ángel Baldán, Oleg A. Andreev, Diego Rodríguez-Puyol, Raman Bahal, Yana K. Reshetnyak, Yajaira Suárez, Carlos Fernández-Hernando, and Santiago Lamas
JCI Insight, eISSN: 23793708, Published: 15 October 2019 American Society for Clinical Investigation
Previous work has reported the important links between cellular bioenergetics and the development of chronic kidney disease, highlighting the potential for targeting metabolic functions to regulate disease progression. More recently, it has been shown that alterations in fatty acid oxidation (FAO) can have an important impact on the progression of kidney disease. In this work, we demonstrate that loss of miR-33, an important regulator of lipid metabolism, can prevent the repression of FAO in fibrotic kidneys and reduce lipid accumulation. These changes were associated with a dramatic reduction in the extent of fibrosis induced in two different mouse models of kidney disease. These effects were not related to changes in circulating leukocytes, as bone marrow transplant from miR-33 deficient animals did not have a similar impact on disease progression. Most importantly, targeted delivery of miR-33 peptide nucleic acid (PNA) inhibitors to the kidney and other acidic microenvironments was accomplished using pH low insertion peptides (pHLIP) as a carrier. This was effective at both increasing the expression of factors involved in FAO and reducing the development of fibrosis. Together, these findings suggest that miR-33 may be an attractive therapeutic target for the treatment of chronic kidney disease.
Yunpeng Ye, Jakub Toczek, Kiran Gona, Hye-Yeong Kim, Jinah Han, Mahmoud Razavian, Reza Golestani, Jiasheng Zhang, Terence L. Wu, Mousumi Ghosh, Jae-Joon Jung, and Mehran M. Sadeghi
Scientific Reports, eISSN: 20452322, Published: 1 December 2018 Springer Science and Business Media LLC
Matrix metalloproteinases (MMPs) are involved in tissue remodeling. Accordingly, MMP inhibitors and related radiolabeled analogs are important tools for MMP-targeted imaging and therapy in a number of diseases. Herein, we report design, synthesis, and evaluation of a new Arginine-containing macrocyclic hydroxamate analog, RYM, its hydrazinonicotinamide conjugate, RYM1 and 99mTc-labeled analog 99mTc-RYM1 for molecular imaging. RYM exhibited potent inhibition against a panel of recombinant human (rh) MMPs in vitro. RYM1 was efficiently labeled with 99mTcO4− to give 99mTc-RYM1 in a high radiochemical yield and high radiochemical purity. RYM1 and its decayed labeling product displayed similar inhibition potencies against rhMMP-12. Furthermore, 99mTc-RYM1 exhibited specific binding with lung tissue from lung-specific interleukin-13 transgenic mice, in which MMP activity is increased in conjunction with tissue remodeling and inflammation. The results support further development of such new water-soluble Arginine-containing macrocyclic hydroxamate MMP inhibitors for targeted imaging and therapy.
Jakub Toczek, Yunpeng Ye, Kiran Gona, Hye-Yeong Kim, Jinah Han, Mahmoud Razavian, Reza Golestani, Jiasheng Zhang, Terence L. Wu, Jae-Joon Jung, and Mehran M. Sadeghi
Journal of Nuclear Medicine, ISSN: 01615505, eISSN: 2159662X, Pages: 1318-1323, Published: 1 August 2017 Society of Nuclear Medicine
Matrix metalloproteinases (MMPs) play a key role in abdominal aortic aneurysm (AAA) development. Accordingly, MMP-targeted imaging provides important information regarding vessel wall biology in the course of aneurysm development. Given the small size of the vessel wall and its proximity with blood, molecular imaging of aneurysm optimally requires highly sensitive tracers with rapid blood clearance. To this end, we developed a novel hydrosoluble zwitterionic MMP inhibitor, RYM, on the basis of which a pan-MMP tracer, RYM1, was designed. Here, we describe the development and preclinical evaluation of RYM1 in comparison with RP805, a commonly used pan-MMP tracer in murine models of aneurysm. Methods: The macrocyclic hydroxamate-based pan-MMP inhibitor coupled with 6-hydrazinonicotinamide, RYM1, was synthesized and labeled with 99mTc. Radiochemical stability of 99mTc-RYM1 was evaluated by radio–high-performance liquid chromatography analysis. Tracer blood kinetics and biodistribution were compared with 99mTc-RP805 in C57BL/6J mice (n = 10). 99mTc-RYM1 binding to aneurysm and specificity were evaluated by quantitative autoradiography in apolipoprotein E–deficient (apoE−/−) mice with CaCl2-induced carotid aneurysm (n = 11). Angiotensin II–infused apoE−/− (n = 16) mice were used for small-animal SPECT/CT imaging. Aortic tissue MMP activity and macrophage marker CD68 expression were assessed by zymography and reverse-transcription polymerase chain reaction. Results: RYM1 showed nanomolar range inhibition constants for several MMPs. 99mTc-RYM1 was radiochemically stable in mouse blood for 5 h and demonstrated rapid renal clearance and lower blood levels in vivo compared with 99mTc-RP805. 99mTc-RYM1 binding to aneurysm and its specificity were shown by autoradiography in carotid aneurysm. Angiotensin II infusion in apoE−/− mice for 4 wk resulted in AAA formation in 36% (4/11) of surviving animals. In vivo 99mTc-RYM1 small-animal SPECT/CT images showed higher uptake of the tracer in AAA than nondilated aortae. Finally, aortic uptake of 99mTc-RYM1 in vivo correlated with aortic MMP activity and CD68 expression. Conclusion: The newly developed pan-MMP inhibitor–based tracer 99mTc-RYM1 displays favorable pharmacokinetics for early vascular imaging and enables specific detection of inflammation and MMP activity in aneurysm.
Sina Tavakoli, Kevin Downs, John D. Short, Huynh Nga Nguyen, Yanlai Lai, Paul A. Jerabek, Beth Goins, Jakub Toczek, Mehran M. Sadeghi, and Reto Asmis
Arteriosclerosis, Thrombosis, and Vascular Biology, ISSN: 10795642, eISSN: 15244636, Pages: 1840-1848, Published: 2017 Ovid Technologies (Wolters Kluwer Health)
Objective— Despite the early promising results of 18F-fluorodeoxyglucose positron emission tomography for assessment of vessel wall inflammation, its accuracy in prospective identification of vulnerable plaques has remained limited. Additionally, previous studies have indicated that 18F-fluorodeoxyglucose uptake alone may not allow for accurate identification of specific macrophage activation states. We aimed to determine whether combined measurement of glucose and glutamine accumulation—the 2 most important bioenergetic substrates for macrophages—improves the distinction of macrophage inflammatory states and can be utilized to image atherosclerosis. Approach and Results— Murine peritoneal macrophages (M&PHgr;) were activated ex vivo into proinflammatory states with either lipopolysaccharide (M&PHgr;LPS) or interferon-&ggr;+tumor necrosis factor-&agr; (M&PHgr;IFN-&ggr;+TNF-&agr;). An alternative polarization phenotype was induced with interleukin-4 (M&PHgr;IL-4). The pronounced increase in 2-deoxyglucose uptake distinguishes M&PHgr;LPS from M&PHgr;IFN-&ggr;+TNF-&agr;, M&PHgr;IL-4, and unstimulated macrophages (M&PHgr;0). Despite having comparable levels of 2-deoxyglucose accumulation, M&PHgr;IL-4 can be distinguished from both M&PHgr;IFN-&ggr;+TNF-&agr; and M&PHgr;0 based on the enhanced glutamine accumulation, which was associated with increased expression of a glutamine transporter, Slc1a5. Ex vivo autoradiography experiments demonstrated distinct and heterogenous patterns of 18F-fluorodeoxyglucose and 14C-glutamine accumulation in atherosclerotic lesions of low-density lipoprotein receptor-null mice fed a high-fat diet. Conclusions— Combined assessment of glutamine and 2-deoxyglucose accumulation improves the ex vivo identification of macrophage activation states. Combined ex vivo metabolic imaging demonstrates heterogenous and distinct patterns of substrate accumulation in atherosclerotic lesions. Further studies are required to define the in vivo significance of glutamine uptake in atherosclerosis and its potential application in identification of vulnerable plaques.
Reza Golestani, Mahmoud Razavian, Yunpeng Ye, Jiasheng Zhang, Jae-Joon Jung, Jakub Toczek, Kiran Gona, Hye-Yeong Kim, Jack A. Elias, Chun Geun Lee, Robert J. Homer, and Mehran M. Sadeghi
Journal of Nuclear Medicine, ISSN: 01615505, eISSN: 2159662X, Pages: 138-143, Published: 1 January 2017 Society of Nuclear Medicine
Imaging techniques for detection of molecular and cellular processes that precede or accompany lung diseases are needed. Matrix metalloproteinases (MMPs) play key roles in the development of pulmonary pathology. The objective of this study was to investigate the feasibility of in vivo MMP-targeted molecular imaging for detection of lung inflammation and remodeling. Methods: Lung-specific IL-13 transgenic (Club cell 10-kDa protein [CC10]-IL-13 Tg) mice and wild-type littermates were used in this study. Lung structure, gene expression, and MMP activity were assessed by histology, real-time reverse transcription polymerase chain reaction, Western blotting, and zymography. MMP activation was imaged by in vivo small-animal SPECT/CT followed by ex vivo planar imaging. Signal specificity was addressed using a control tracer. The correlation between in vivo MMP signal and gene expression was addressed. Results: CC10-IL-13 Tg mice developed considerable pulmonary tissue remodeling and inflammation. CD68, MMP-12, and MMP-13 were significantly higher in CC10-IL-13 Tg lungs. On in vivo small-animal SPECT/CT and ex vivo planar images, the MMP signal was significantly higher in the lungs of CC10-IL-13 Tg mice than wild-type animals. Furthermore, a nonbinding analog tracer showed significantly lower accumulation in CC10-IL-13 Tg lungs relative to the specific tracer. There was a significant correlation between small-animal SPECT/CT–derived MMP signal and CD68 expression in the lungs (r = 0.70, P < 0.01). Conclusion: Small-animal SPECT/CT–based MMP-targeted imaging of the lungs is feasible and reflects pulmonary inflammation. If validated in humans, molecular imaging of inflammation and remodeling can potentially help early diagnosis and monitoring of the effects of therapeutic interventions in pulmonary diseases.
Mahmoud Razavian, Thomas Bordenave, Dimitris Georgiadis, Fabrice Beau, Jiasheng Zhang, Reza Golestani, Jakub Toczek, Jae-Joon Jung, Yunpeng Ye, Hye-Yeong Kim, Jinah Han, Vincent Dive, Laurent Devel, and Mehran M. Sadeghi
Scientific Reports, eISSN: 20452322, Published: 5 December 2016 Springer Science and Business Media LLC
Matrix metalloproteinase (MMP)-12 plays a key role in the development of aneurysm. Like other members of MMP family, MMP-12 is produced as a proenzyme, mainly by macrophages, and undergoes proteolytic activation to generate an active form. Accordingly, molecular imaging of the MMP-12 active form can inform of the pathogenic process in aneurysm. Here, we developed a novel family of fluorescent probes based on a selective MMP-12 inhibitor, RXP470.1 to target the active form of MMP-12. These probes were stable in complex media and retained the high affinity and selectivity of RXP470.1 for MMP-12. Amongst these, probe 3 containing a zwitterionic fluorophore, ZW800-1, combined a favorable affinity profile toward MMP-12 and faster blood clearance. In vivo binding of probe 3 was observed in murine models of sterile inflammation and carotid aneurysm. Binding specificity was demonstrated using a non-binding homolog. Co-immunostaining localized MMP-12 probe binding to MMP-12 positive areas and F4/80 positive macrophages in aneurysm. In conclusion, the active form of MMP-12 can be detected by optical imaging using RXP470.1-based probes. This is a valuable adjunct for pathophysiology research, drug development, and potentially clinical applications.
Thomas Bordenave, Marion Helle, Fabrice Beau, Dimitris Georgiadis, Livia Tepshi, Mylène Bernes, Yunpeng Ye, Laure Levenez, Enora Poquet, Hervé Nozach, Mahmoud Razavian, Jakub Toczek, Enrico A. Stura, Vincent Dive, Mehran M. Sadeghi, and Laurent Devel
Bioconjugate Chemistry, ISSN: 10431802, eISSN: 15204812, Pages: 2407-2417, Published: 19 October 2016 American Chemical Society (ACS)
In designing new tracers consisting of a small peptide conjugated to a reporter of comparable size, particular attention needs to be paid to the selection of the reporter group, which can dictate both the in vitro and the in vivo performances of the whole conjugate. In the case of fluorescent tracers, this is particularly true given the large numbers of available dye moieties differing in their structures and properties. Here, we have investigated the in vitro and in vivo properties of a novel series of MMP-12 selective probes composed of cyanine dyes varying in their structure, net charge, and hydrophilic character, tethered through a linker to a potent and specific MMP-12 phosphinic pseudopeptide inhibitor. The impact of linker length has been also explored. The crystallographic structure of one tracer in complex with MMP-12 has been obtained, providing the first crystal structure of a Cy5.5-derived probe and confirming that the binding of the targeting moiety is unaffected. MMP-12 remains the tracers' privileged target, as attested by their affinity selectivity profile evaluated in solution toward a panel of 12 metalloproteases. In vivo assessment of four selected probes has highlighted not only the impact of the dye structure but also that of the linker length on the probes' blood clearance rates and their biodistributions. These experiments have also provided valuable data on the stability of the dye moieties in vivo. This has permitted the identification of one probe, which combines favorable binding to MMP-12 in solution and on cells with optimized in vivo performance including blood clearance rate suitable for short-time imaging. Through this series of tracers, we have identified various critical factors modulating the tracers' in vivo behavior, which is both useful for the development and optimization of MMP-12 selective radiolabeled tracers and informative for the design of fluorescent probes in general.
Jae-Joon Jung, Mahmoud Razavian, Hye-Yeong Kim, Yunpeng Ye, Reza Golestani, Jakub Toczek, Jiasheng Zhang, and Mehran M. Sadeghi
Scientific Reports, eISSN: 20452322, Published: 13 September 2016 Springer Science and Business Media LLC
Calcific aortic valve disease (CAVD) is the most common cause of aortic stenosis. Currently, there is no non-invasive medical therapy for CAVD. Matrix metalloproteinases (MMPs) are upregulated in CAVD and play a role in its pathogenesis. Here, we evaluated the effect of doxycycline, a nonselective MMP inhibitor on CAVD progression in the mouse. Apolipoprotein (apo)E−/− mice (n = 20) were fed a Western diet (WD) to induce CAVD. After 3 months, half of the animals was treated with doxycycline, while the others continued WD alone. After 6 months, we evaluated the effect of doxycycline on CAVD progression by echocardiography, MMP-targeted micro single photon emission computed tomography (SPECT)/computed tomography (CT), and tissue analysis. Despite therapeutic blood levels, doxycycline had no significant effect on MMP activation, aortic valve leaflet separation or flow velocity. This lack of effect on in vivo images was confirmed on tissue analysis which showed a similar level of aortic valve gelatinase activity, and inflammation between the two groups of animals. In conclusion, doxycycline (100 mg/kg/day) had no effect on CAVD progression in apoE−/− mice with early disease. Studies with more potent and specific inhibitors are needed to establish any potential role of MMP inhibition in CAVD development and progression.
Jakub Toczek and Mehran M. Sadeghi
Circulation: Cardiovascular Imaging, ISSN: 19419651, eISSN: 19420080, Published: 1 May 2016 Ovid Technologies (Wolters Kluwer Health)
Carotid artery disease accounts for ≈20% of strokes.1,2 Although the role of revascularization in reducing the incidence of stroke in symptomatic carotid stenosis is well established,1 the optimal treatment for asymptomatic disease remains controversial.2,3 In Asymptomatic Carotid Atherosclerosis Study (ACAS, published in 1995)4 and Asymptomatic Carotid Surgery Trial 1 (ACST-1, published in 2004),5 which enrolled patients with ≥60% carotid stenosis carotid endarterectomy (CEA) was found to be superior to medical therapy. Subsequent studies demonstrated that carotid stenting, the alternative revascularization procedure, is not inferior to CEA in these patients.6,7 However, recent advances in medical therapy have led to considerable reduction in stroke risk, such that the contemporary annual rate of ipsilateral stroke in medically managed patients with asymptomatic carotid stenosis (≤1%) is similar to the risks observed with revascularization in aforementioned trials.2 Accordingly, American Heart Association guidelines suggest that revascularization may be considered in a highly selected group of patients with asymptomatic carotid stenosis, acknowledging that the effectiveness of this approach compared to medical therapy is not well established.8 How to identify this high-risk group of patients remains more akin to an art than evidence-based medicine. Interestingly, in contrast to Canada and most of Europe, a great majority of carotid revascularization procedures in the United States are performed on asymptomatic patients,2 highlighting the magnitude of the problem and costs associated with potentially unnecessary procedures. See Article by Voo et al Focal thrombosis triggered by plaque rupture, superficial erosion, or protruding calcified nodule in the setting of predisposing systemic factors can be asymptomatic or lead to myocardial infarction and stroke. Plaque rupture is the main cause of such thrombotic complications in both coronary and carotid artery disease. The nidus for rupture, a thin-cap, highly inflammatory fibroatheroma …
Jakub Toczek and Mehran M. Sadeghi
Journal of Nuclear Cardiology, ISSN: 10713581, eISSN: 15326551, Pages: 271-273, Published: 1 April 2016 Springer Science and Business Media LLC
The purpose of cardiovascular molecular imaging is to detect, characterize, and quantify relevant pathobiological processes at molecular and cellular levels in humans and other living systems. Thus, molecular imaging can be perceived best in contrast with anatomical imaging, where the focus is on the structure (as opposed to a process), and imaging of tissues or cells outside of the living systems. By this definition, cardiac CT to detect coronary calcification is not a molecular imaging technique, but F-NaF PET imaging of the calcification process in coronary arteries is a prototypical example of cardiovascular molecular imaging. These boundaries are somewhat blurry regarding physiological imaging. The quantification of left ventricular ejection fraction using various imaging modalities is clearly outside the realm of molecular imaging. However, whether or not to classify other examples of physiological imaging as molecular imaging can be debatable. As such, myocardial perfusion imaging is considered by many as an early example of molecular imaging in cardiovascular medicine. For the sake of clarity, we prefer to reserve the term ‘‘molecular imaging’’ for techniques that target cellular and molecular processes directly. With this narrower definition, F-fluorodeoxyglucose imaging of myocardial metabolism is molecular imaging, while myocardial perfusion imaging is not. Fundamentally, cardiovascular molecular imaging is multidisciplinary. Related fields range from clinical cardiovascular medicine (identification of a diagnostic gap) to biology (identification of potential targets), chemistry (generation of ligands and development of molecular imaging probes), physics (instrumentation, probe contrast detection and signal processing) and data processing (Figure 1). A comprehensive insight into these components is required for meaningful analysis of molecular imaging data, at least in the early stages of development, and their validation prior to translation form preclinical studies to clinical trials and ultimately clinical practice. The promise of molecular imaging is in personalized, precision, medicine, and improving patient care by addressing diagnostic gaps that traditional tests have
Jakub Toczek, Judith L. Meadows, and Mehran M. Sadeghi
Circulation: Cardiovascular Imaging, ISSN: 19419651, eISSN: 19420080, Published: 1 January 2016 Ovid Technologies (Wolters Kluwer Health)
Selection of patients for abdominal aortic aneurysm repair is currently based on aneurysm size, growth rate, and symptoms. Molecular imaging of biological processes associated with aneurysm growth and rupture, for example, inflammation and matrix remodeling, could improve patient risk stratification and lead to a reduction in abdominal aortic aneurysm morbidity and mortality. 18F-fluorodeoxyglucose-positron emission tomography and ultrasmall superparamagnetic particles of iron oxide magnetic resonance imaging are 2 novel approaches to abdominal aortic aneurysm imaging evaluated in clinical trials. A variety of other tracers, including those that target inflammatory cells and proteolytic enzymes (eg, integrin &agr;v&bgr;3 and matrix metalloproteinases), have proven effective in preclinical models of abdominal aortic aneurysm and show great potential for clinical translation.
A. Broisat, J. Toczek, L. S. Dumas, M. Ahmadi, S. Bacot, P. Perret, L. Slimani, G. Barone-Rochette, A. Soubies, N. Devoogdt, T. Lahoutte, D. Fagret, L. M. Riou, and C. Ghezzi
Journal of Nuclear Medicine, ISSN: 01615505, eISSN: 2159662X, Pages: 1678-1684, Published: 1 October 2014 Society of Nuclear Medicine
99mTc-cAbVCAM1-5, a single-domain antibody fragment directed against mouse or human vascular cell adhesion molecule 1 (VCAM-1), recently has been proposed as a new imaging agent for the detection of inflamed atherosclerotic lesions. Indeed, in a mouse model of atherosclerosis, 99mTc-cAbVCAM1-5 specifically bound to VCAM-1–positive lesions, thereby allowing their identification on SPECT images. The purpose of the present study was to investigate 99mTc-cAbVCAM1-5 imaging sensitivity using a reference statin therapy. Methods: Thirty apolipoprotein E–deficient mice were fed a western-type diet. First, the relationship between the level of VCAM-1 expression and 99mTc-cAbVCAM1-5 uptake was evaluated in 18 mice using immunohistochemistry and autoradiography. Second, longitudinal SPECT/CT imaging was performed on control (n = 9) or atorvastatin-treated mice (0.01% w/w, n = 9). Results: 99mTc-cAbVCAM1-5 uptake in atherosclerotic lesions correlated with the level of VCAM-1 expression (P < 0.05). Atorvastatin exerted significant antiatherogenic effects, and 99mTc-cAbVCAM1-5 lesion uptake was significantly reduced in 35-wk-old atorvastatin-treated mice, as indicated by ex vivo γ-well counting and autoradiography (P < 0.05). SPECT imaging quantification based on contrast-enhanced CT was reproducible (interexperimenter intraclass correlation coefficient, 0.97; intraexperimenter intraclass correlation coefficient, 0.90), and yielded results that were highly correlated with tracer biodistribution (r = 0.83; P < 0.0001). Therefore, reduced 99mTc-cAbVCAM1-5 uptake in atorvastatin-treated mice was successfully monitored noninvasively by SPECT/CT imaging (0.87 ± 0.06 vs. 1.11 ± 0.09 percentage injected dose per cubic centimeter in control group, P < 0.05). Conclusion: 99mTc-cAbVCAM1-5 imaging allowed the specific, sensitive, and reproducible quantification of VCAM-1 expression in mouse atherosclerotic lesions. 99mTc-cAbVCAM1-5 therefore exhibits suitable characteristics for the evaluation of novel antiatherogenic agents.
Jakub Toczek, Alexis Broisat, Pascale Perret, Marie-Dominique Desruet, Daniel Fagret, Laurent M. Riou, and Catherine Ghezzi
PLoS ONE, eISSN: 19326203, Published: 23 July 2014 Public Library of Science (PLoS)
Background [18F]-fluorodeoxyglucose (FDG) has been suggested for the clinical and experimental imaging of inflammatory atherosclerotic lesions. Significant FDG uptake in brown adipose tissue (BAT) has been observed both in humans and mice. The objective of the present study was to investigate the influence of periaortic BAT on apolipoprotein E-deficient (apoE−/−) mouse atherosclerotic lesion imaging with FDG. Methods ApoE−/− mice (36±2 weeks-old) were injected with FDG (12±2 MBq). Control animals (Group A, n = 7) were injected conscious and kept awake at room temperature (24°C) throughout the accumulation period. In order to minimize tracer activity in periaortic BAT, Group B (n = 7) and C (n = 6) animals were injected under anaesthesia at 37°C and Group C animals were additionally pre-treated with propranolol. PET/CT acquisitions were performed prior to animal euthanasia and ex vivo analysis of FDG biodistribution. Results Autoradiographic imaging indicated higher FDG uptake in atherosclerotic lesions than in the normal aortic wall (all groups, P<0.05) and the blood (all groups, P<0.01) which correlated with macrophage infiltration (R = 0.47; P<0.001). However, periaortic BAT uptake was either significantly higher (Group A, P<0.05) or similar (Group B and C, P = NS) to that observed in atherosclerotic lesions and was shown to correlate with in vivo quantified aortic FDG activity. Conclusion Periaortic BAT FDG uptake was identified as a confounding factor while using FDG for the non-invasive imaging of mouse atherosclerotic lesions.
Jacques Ohayon, Nicolas Mesnier, Alexis Broisat, Jakub Toczek, Laurent Riou, and Philippe Tracqui
Biomechanics and Modeling in Mechanobiology, ISSN: 16177959, eISSN: 16177940, Pages: 801-813, Published: July 2012 Springer Science and Business Media LLC
The structure of mouse atherosclerotic lesions may differ from that of humans, and mouse atherosclerotic plaques do not rupture except in some specific locations such as the brachiocephalic artery. Recently, our group was the first to observe that the amplitudes of in vivo stresses in ApoE−/− mouse aortic atherosclerotic lesions were much lower and differed from those found in a previous work performed on human lesions. In this previous preliminary work, we hypothesized that the plaque mechanical properties (MP) may in turn be responsible for such species differences. However, the limited number of human samples used in our previous comparative study was relevant but not sufficient to broadly validate such hypothesis. Therefore, in this study, we propose an original finite element strategy that reconstructs the in vivo stress/strain (IVS/S) distributions in ApoE−/− artherosclerotic vessels based on cross substitution of ApoE−/− mouse and human plaque components stiffnesses and including residual stress/strain (RS/S). Our results: (1) showed that including RS/S decreases by a factor 2 the amplitude of maximal IVS/S, and more importantly, (2) demonstrated that the MP of the ApoE−/− plaque constituents are mainly responsible for the low level—compared with human—of intraplaque stress in ApoE−/− mouse aortic atherosclerotic lesions (8.36 ± 2.63kPa vs. 182.25 ± 55.88kPa for human). Our study highlights that such differences in the distribution and amplitude of vessel wall stress might be one key feature for explaining for the difference in lesion stability between human coronary and mouse aortic lesions.
Alexis Broisat, Sophie Hernot, Jakub Toczek, Jens De Vos, Laurent M. Riou, Sandrine Martin, Mitra Ahmadi, Nicole Thielens, Ulrich Wernery, Vicky Caveliers, Serge Muyldermans, Tony Lahoutte, Daniel Fagret, Catherine Ghezzi, and Nick Devoogdt
Circulation Research, ISSN: 00097330, eISSN: 15244571, Volume: 110, Pages: 927-937, Published: 30 March 2012 Ovid Technologies (Wolters Kluwer Health)
Rationale: A noninvasive tool allowing the detection of vulnerable atherosclerotic plaques is highly needed. By combining nanomolar affinities and fast blood clearance, nanobodies represent potential radiotracers for cardiovascular molecular imaging. Vascular cell adhesion molecule-1 (VCAM1) constitutes a relevant target for molecular imaging of atherosclerotic lesions. Objective: We aimed to generate, radiolabel, and evaluate anti-VCAM1 nanobodies for noninvasive detection of atherosclerotic lesions. Methods and Results: Ten anti-VCAM1 nanobodies were generated, radiolabeled with technetium-99m, and screened in vitro on mouse and human recombinant VCAM1 proteins and endothelial cells and in vivo in apolipoprotein E–deficient (ApoE−/−) mice. A nontargeting control nanobody was used in all experiments to demonstrate specificity. All nanobodies displayed nanomolar affinities for murine VCAM1. Flow cytometry analyses using human human umbilical vein endothelial cells indicated murine and human VCAM1 cross-reactivity for 6 of 10 nanobodies. The lead compound cAbVCAM1-5 was cross-reactive for human VCAM1 and exhibited high lesion-to-control (4.95±0.85), lesion-to-heart (8.30±1.11), and lesion-to-blood ratios (4.32±0.48) (P<0.05 versus control C57Bl/6J mice). Aortic arch atherosclerotic lesions of ApoE−/− mice were successfully identified by single-photon emission computed tomography imaging. 99mTc-cAbVCAM1-5 binding specificity was demonstrated by in vivo competition experiments. Autoradiography and immunohistochemistry further confirmed cAbVCAM1-5 uptake in VCAM1-positive lesions. Conclusions: The 99mTc-labeled, anti-VCAM1 nanobody cAbVCAM1-5 allowed noninvasive detection of VCAM1 expression and displayed mouse and human cross-reactivity. Therefore, this study demonstrates the potential of nanobodies as a new class of radiotracers for cardiovascular applications. The nanobody technology might evolve into an important research tool for targeted imaging of atherosclerotic lesions and has the potential for fast clinical translation.
Sophie Hernot, Sunil Unnikrishnan, Zhongmin Du, Talent Shevchenko, Bernard Cosyns, Alexis Broisat, Jakub Toczek, Vicky Caveliers, Serge Muyldermans, Tony Lahoutte, Alexander L. Klibanov, and Nick Devoogdt
Journal of Controlled Release, ISSN: 01683659, eISSN: 18734995, Volume: 158, Pages: 346-353, Published: 10 March 2012 Elsevier BV
Camelid-derived single-domain antibody-fragments (~15kDa), called nanobodies, are a new class of molecular tracers that are routinely identified with nanomolar affinity for their target and that are easily tailored for molecular imaging and drug delivery applications. We hypothesized that they are well-suited for the design of targeted microbubbles (μBs) and aimed to develop and characterize eGFP- and VCAM-1-targeted μBs. Anti-eGFP (cAbGFP4) and anti-VCAM-1 (cAbVCAM1-5) nanobodies were site-specifically biotinylated in bacteria. This metabolic biotinylation method yielded functional nanobodies with one biotin located at a distant site of the antigen-binding region of the molecule. The biotinylated nanobodies were coupled to biotinylated lipid μBs via streptavidin-biotin bridging. The ability of μB-cAbGFP4 to recognize eGFP was tested as proof-of-principle by fluorescent microscopy and confirmed the specific binding of eGFP to μB-cAbGFP4. Dynamic flow chamber studies demonstrated the ability of μB-cAbVCAM1-5 to bind VCAM-1 in fast flow (up to 5 dynes/cm(2)). In vivo targeting studies were performed in MC38 tumor-bearing mice (n=4). μB-cAbVCAM1-5 or control μB-cAbGFP4 were injected intravenously and imaged using a contrast-specific ultrasound imaging mode. The echo intensity in the tumor was measured 10min post-injection. μB-cAbVCAM1-5 showed an enhanced signal compared to control μBs (p<0.05). Using metabolic and site-specific biotinylation of nanobodies, a method to develop nanobody-coupled μBs was described. The application of VCAM-1-targeted μBs as novel molecular ultrasound contrast agent was demonstrated both in vitro and in vivo.