Collagen Hybridizing Peptide–Based Radiotracers for Molecular Imaging of Collagen Turnover in Pulmonary Fibrosis Azmi A. Ahmad, Mean Ghim, Gunjan Kukreja, Afarin Neishabouri, Zhengxing Zhang, et al. Journal of Nuclear Medicine, 2025 Pulmonary fibrosis is a characteristic feature of interstitial lung disease. Current clinical diagnostic methods provide a snapshot of the lung structure without information on disease activity. Collagen hybridizing peptides offer the opportunity to detect collagen remodeling through their hybridization with denatured collagen. Here, we sought to develop a 99mTc-labeled collagen hybridizing tracer to track denatured collagen in vivo and validate it in a murine model of pulmonary fibrosis. Methods: Imaging agents consisting of a polyhistidine or a poly-histidine-glutamic acid [(HE)3] peptide connected to an N-terminal targeting moiety with 9 glycine-proline-hydroxyproline repeats [(GPO)9] through a 3-glycine linker were synthesized. After radiolabeling with 99mTc-tricarbonyl, the labeled products' purity and stability were evaluated by high-performance liquid chromatography and γ-well counting, and their biodistributions were compared in mice. To induce pulmonary fibrosis, the lungs of 8- to 10-wk-old mice were exposed to bleomycin (or saline as control). At 3 wk after induction, SPECT/CT imaging with 99mTc-(HE)3-(GPO)9 was performed 1 h after injection and was followed by tissue collection to assess 99mTc-(HE)3-(GPO)9 biodistribution by γ-well counting and to evaluate lung histology. The specificity of the tracer uptake was assessed using a scrambled homolog. A group of animals underwent serial imaging 3 and 8-10 wk after induction. Results: The specific activity of the final radiolabeled product was 70.3 ± 14.8 GBq/µmol. Radiolabeled tracers were stable in blood for at least 2 h and showed rapid blood clearance. 99mTc-(HE)3-(GPO)9 showed lower liver uptake in biodistribution studies and was selected for in vivo imaging studies. SPECT/CT imaging of bleomycin-treated mice 3 wk after induction showed higher specific 99mTc-(HE)3-(GPO)9 lung uptake than that of control mice (P < 0.01) and that of bleomycin-treated mice 8-10 wk after induction, when fibrosis was resolved (P < 0.05). There was a significant correlation between lung uptake quantified by SPECT/CT and γ-well counting (Pearson R = 0.83, P < 0.001) and significant correlations between tracer uptake and indices of tissue fibrosis. Conclusion: 99mTc-(HE)3-(GPO)9 enables SPECT imaging of collagen turnover in pulmonary fibrosis. This approach expands the scope of existing diagnostic tools in fibrosis and can lead to better patient management by monitoring the effect of antifibrotic therapies.
Homeostatic, Non-Canonical Role of Macrophage Elastase in Vascular Integrity Mani Salarian, Mean Ghim, Jakub Toczek, Jinah Han, Dar Weiss, et al. Circulation Research, 2023 Background: Matrix metalloproteinase (MMP)-12 is highly expressed in abdominal aortic aneurysms and its elastolytic function has been implicated in the pathogenesis. This concept is challenged, however, by conflicting data. Here, we sought to revisit the role of MMP-12 in abdominal aortic aneurysm. Methods: Apoe -/- and Mmp12 -/- / Apoe -/- mice were infused with Ang II (angiotensin). Expression of neutrophil extracellular traps (NETs) markers and complement component 3 (C3) levels were evaluated by immunostaining in aortas of surviving animals. Plasma complement components were analyzed by immunoassay. The effects of a complement inhibitor, IgG-FH 1-5 (factor H-immunoglobulin G), and macrophage-specific MMP-12 deficiency on adverse aortic remodeling and death from rupture in Ang II–infused mice were determined. Results: Unexpectedly, death from aortic rupture was significantly higher in Mmp12 -/- / Apoe -/- mice. This associated with more neutrophils, citrullinated histone H3 and neutrophil elastase, markers of NETs, and C3 levels in Mmp12 -/- aortas. These findings were recapitulated in additional models of abdominal aortic aneurysm. MMP-12 deficiency also led to more pronounced elastic laminae degradation and reduced collagen integrity. Higher plasma C5a in Mmp12 -/- mice pointed to complement overactivation. Treatment with IgG-FH 1-5 decreased aortic wall NETosis and reduced adverse aortic remodeling and death from rupture in Ang II–infused Mmp12 -/- mice. Finally, macrophage-specific MMP-12 deficiency recapitulated the effects of global MMP-12 deficiency on complement deposition and NETosis, as well as adverse aortic remodeling and death from rupture in Ang II–infused mice. Conclusions: An MMP-12 deficiency/complement activation/NETosis pathway compromises aortic integrity, which predisposes to adverse vascular remodeling and abdominal aortic aneurysm rupture. Considering these new findings, the role of macrophage MMP-12 in vascular homeostasis demands re-evaluation of MMP-12 function in diverse settings.
Multimodality Imaging of Aortic Valve Calcification and Function in a Murine Model of Calcific Aortic Valve Disease and Bicuspid Aortic Valve Azmi A. Ahmad, Mean Ghim, Jakub Toczek, Afarin Neishabouri, Devi Ojha, et al. Journal of Nuclear Medicine, 2023 Visual Abstract Calcific aortic valve disease (CAVD) is a prevailing disease with increasing occurrence and no known medical therapy. Dcbld2−/− mice have a high prevalence of bicuspid aortic valve (BAV), spontaneous aortic valve calcification, and aortic stenosis (AS). 18F-NaF PET/CT can detect the aortic valve calcification process in humans. However, its feasibility in preclinical models of CAVD remains to be determined. Here, we sought to validate 18F-NaF PET/CT for tracking murine aortic valve calcification and leveraged it to examine the development of calcification with aging and its interdependence with BAV and AS in Dcbld2−/− mice. Methods: Dcbld2−/− mice at 3–4 mo, 10–16 mo, and 18–24 mo underwent echocardiography, 18F-NaF PET/CT (n = 34, or autoradiography (n = 45)), and tissue analysis. A subset of mice underwent both PET/CT and autoradiography (n = 12). The aortic valve signal was quantified as SUVmax on PET/CT and as percentage injected dose per square centimeter on autoradiography. The valve tissue sections were analyzed by microscopy to identify tricuspid and bicuspid aortic valves. Results: The aortic valve 18F-NaF signal on PET/CT was significantly higher at 18–24 mo (P < 0.0001) and 10–16 mo (P < 0.05) than at 3–4 mo. Additionally, at 18–24 mo BAV had a higher 18F-NaF signal than tricuspid aortic valves (P < 0.05). These findings were confirmed by autoradiography, with BAV having significantly higher 18F-NaF uptake in each age group. A significant correlation between PET and autoradiography data (Pearson r = 0.79, P < 0.01) established the accuracy of PET quantification. The rate of calcification with aging was significantly faster for BAV (P < 0.05). Transaortic valve flow velocity was significantly higher in animals with BAV at all ages. Finally, there was a significant correlation between transaortic valve flow velocity and aortic valve calcification by both PET/CT (r = 0.55, P < 0.001) and autoradiography (r = 0.45, P < 0.01). Conclusion: 18F-NaF PET/CT links valvular calcification to BAV and aging in Dcbld2−/− mice and suggests that AS may promote calcification. In addition to addressing the pathobiology of valvular calcification, 18F-NaF PET/CT may be a valuable tool for evaluation of emerging therapeutic interventions in CAVD.
Positron Emission Tomography Imaging of Vessel Wall Matrix Metalloproteinase Activity in Abdominal Aortic Aneurysm Jakub Toczek, Kiran Gona, Yongjian Liu, Azmi Ahmad, Mean Ghim, et al. Circulation Cardiovascular Imaging, 2023 Background: Matrix metalloproteinases (MMPs) play a key role in the pathogenesis of abdominal aortic aneurysm (AAA). Imaging aortic MMP activity, especially using positron emission tomography to access high sensitivity, quantitative data, could potentially improve AAA risk stratification. Here, we describe the design, synthesis, characterization, and evaluation in murine AAA and human aortic tissue of a first-in-class MMP-targeted positron emission tomography radioligand, 64 Cu-RYM2. Methods: The broad spectrum MMP inhibitor, RYM2 was synthetized, and its potency as an MMP inhibitor was evaluated by a competitive inhibition assay. Toxicology studies were performed. Tracer biodistribution was evaluated in a murine model of AAA induced by angiotensin II infusion in Apolipoprotein E-deficient mice. 64 Cu-RYM2 binding to normal and aneurysmal human aortic tissues was assessed by autoradiography. Results: RYM2 functioned as an MMP inhibitor with nanomolar affinities. Toxicology studies showed no adverse reaction in mice. Upon radiolabeling with Cu-64, the resulting tracer was stable in murine and human blood in vitro. Biodistribution and metabolite analysis in mice showed rapid renal clearance and acceptable in vivo stability. In vivo positron emission tomography/computed tomography in a murine model of AAA showed a specific aortic signal, which correlated with ex vivo measured MMP activity and Cd68 gene expression. 64 Cu-RYM2 specifically bound to normal and aneurysmal human aortic tissues in correlation with MMP activity. Conclusions: 64 Cu-RYM2 is a first-in-class MMP-targeted positron emission tomography tracer with favorable stability, biodistribution, performance in preclinical AAA, and importantly, specific binding to human tissues. These data set the stage for 64 Cu-RYM2-based translational imaging studies of vessel wall MMP activity, and indirectly, inflammation, in AAA.
Targeted Suppression of miRNA-33 Using pHLIP Improves Atherosclerosis Regression Xinbo Zhang, Noemi Rotllan, Alberto Canfrán-Duque, Jonathan Sun, Jakub Toczek, et al. Circulation Research, 2022 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.
Molecular Imaging of Collagen Turnover in Myocardial Infarction A Neishabouri, M Ghim, O Varli, AA Ahmad, G Kukreja, Z Zhang, J Li, ... Journal of Nuclear Medicine , 2026 2026
Aging, matrix metalloproteinase imaging, and survival prospects in aortic aneurysm M Ghim, O Varli, AA Ahmad, A Neishabouri, G Kukreja, Z Zhang, ... bioRxiv , 2026 2026
Correlates of denatured collagen in myocardial infarction A Neishabouri, M Ghim, A Ahmad, G Kukreja, Z Zhang, J Li, M Salarian, ... Journal of Nuclear Medicine 66 (supplement 1), 252190-252190 , 2025 2025
Collagen hybridizing peptide–based radiotracers for molecular imaging of collagen turnover in pulmonary fibrosis AA Ahmad, M Ghim, G Kukreja, A Neishabouri, Z Zhang, J Li, M Salarian, ... Journal of Nuclear Medicine 66 (3), 425-433 , 2025 2025 Citations: 5
Compounds For Molecular Imaging of Collagen Turnover and Methods Using Same M Sadeghi, M SALARIAN, K Gona, J TOCZEK US Patent App. 18/573,604 , 2024 2024
Molecular imaging of collagen remodeling in a murine model of lung fibrosis A Ahmad, M Ghim, G Kukreja, A Neishabouri, Z Zhang, J Li, M Salarian, ... Journal of Nuclear Medicine 65 (supplement 2), 242026-242026 , 2024 2024 Citations: 1
Multimodality imaging of aortic valve calcification and function in a murine model of calcific aortic valve disease and bicuspid aortic valve AA Ahmad, M Ghim, J Toczek, A Neishabouri, D Ojha, Z Zhang, K Gona, ... Journal of Nuclear Medicine 64 (9), 1487-1494 , 2023 2023 Citations: 8
Homeostatic, non-canonical role of macrophage elastase in vascular integrity M Salarian, M Ghim, J Toczek, J Han, D Weiss, B Spronck, ... Circulation research 132 (4), 432-448 , 2023 2023 Citations: 34
Positron emission tomography imaging of vessel wall matrix metalloproteinase activity in abdominal aortic aneurysm J Toczek, K Gona, Y Liu, A Ahmad, M Ghim, D Ojha, G Kukreja, ... Circulation: Cardiovascular Imaging 16 (1), e014615 , 2023 2023 Citations: 14
PET Imaging of Matrix Metalloproteinase Activity in Abdominal Aortic Aneurysm; Toward Clinical Translation K Gona, J Toczek, A Ahmad, M Ghim, D Ojha, M Salarian, R Guzman, ... Journal of Nuclear Medicine 63 (supplement 2), 2218-2218 , 2022 2022
Deletion of matrix metalloproteinase-12 compromises mechanical homeostasis and leads to an aged aortic phenotype in young mice B Spronck, AB Ramachandra, L Moriyama, J Toczek, J Han, MM Sadeghi, ... Journal of biomechanics 141, 111179 , 2022 2022 Citations: 5
Targeted suppression of miRNA-33 using pHLIP improves atherosclerosis regression X Zhang, N Rotllan, A Canfrán-Duque, J Sun, J Toczek, A Moshnikova, ... Circulation Research 131 (1), 77-90 , 2022 2022 Citations: 87
Matrix Metalloproteinase Inhibitors and Imaging Agents, And Methods Using Same M Sadeghi, Y Ye, KIM Hye-Yeong, HY HUANG, J TOCZEK US Patent App. 17/693,011 , 2022 2022
Differential BMP signaling mediates the interplay between genetics and leaflet numbers in aortic valve calcification JJ Jung, AA Ahmad, S Rajendran, L Wei, J Zhang, J Toczek, L Nie, ... Basic to Translational Science 7 (4), 333-345 , 2022 2022 Citations: 13
Considerations on PET/MR imaging of carotid plaque inflammation with 68 Ga-Pentixafor J Toczek, L Riou Journal of Nuclear Cardiology 29 (2), 503-505 , 2022 2022 Citations: 4
Matrix metalloproteinase inhibitors and imaging agents, and methods using same M Sadeghi, Y Ye, KIM Hye-Yeong, HY HUANG, J TOCZEK US Patent 11,286,251 , 2022 2022
Evolution of arterial [ 18 F]-sodium fluoride uptake and calcification J Toczek Journal of Nuclear Cardiology 28 (5), 1946-1948 , 2021 2021
Identifying the leukocyte uptake pattern of inflammation imaging agents: Current limitations and potential impact L Riou, J Toczek, A Broisat, C Ghezzi, L Djaileb Journal of Nuclear Cardiology 28 (4), 1646-1648 , 2021 2021 Citations: 1
Computed tomography imaging of macrophage phagocytic activity in abdominal aortic aneurysm J Toczek, P Boodagh, N Sanzida, M Ghim, M Salarian, K Gona, G Kukreja, ... Theranostics 11 (12), 5876 , 2021 2021 Citations: 16
FDG PET imaging of vascular inflammation in post-traumatic stress disorder: A pilot case–control study J Toczek, AT Hillmer, J Han, C Liu, D Peters, H Emami, J Wu, I Esterlis, ... Journal of Nuclear Cardiology 28 (2), 688-694 , 2021 2021 Citations: 23
MOST CITED SCHOLAR PUBLICATIONS
Nanobodies targeting mouse/human VCAM1 for the nuclear imaging of atherosclerotic lesions A Broisat, S Hernot, J Toczek, J De Vos, LM Riou, S Martin, M Ahmadi, ... Circulation research 110 (7), 927-937 , 2012 2012 Citations: 253
Mapping elasticity moduli of atherosclerotic plaque in situ via atomic force microscopy P Tracqui, A Broisat, J Toczek, N Mesnier, J Ohayon, L Riou Journal of structural biology 174 (1), 115-123 , 2011 2011 Citations: 171
Nanobody-coupled microbubbles as novel molecular tracer S Hernot, S Unnikrishnan, Z Du, T Shevchenko, B Cosyns, A Broisat, ... Journal of controlled release 158 (2), 346-353 , 2012 2012 Citations: 131
Targeted suppression of miRNA-33 using pHLIP improves atherosclerosis regression X Zhang, N Rotllan, A Canfrán-Duque, J Sun, J Toczek, A Moshnikova, ... Circulation Research 131 (1), 77-90 , 2022 2022 Citations: 87
Genetic deficiency or pharmacological inhibition of miR-33 protects from kidney fibrosis NL Price, V Miguel, W Ding, AK Singh, S Malik, N Rotllan, A Moshnikova, ... JCI insight 4 (22), e131102 , 2019 2019 Citations: 77
Characterization of macrophage polarization states using combined measurement of 2-deoxyglucose and glutamine accumulation: implications for imaging of atherosclerosis S Tavakoli, K Downs, JD Short, HN Nguyen, Y Lai, PA Jerabek, B Goins, ... Arteriosclerosis, thrombosis, and vascular biology 37 (10), 1840-1848 , 2017 2017 Citations: 72
99mTc-cAbVCAM1-5 imaging is a sensitive and reproducible tool for the detection of inflamed atherosclerotic lesions in mice A Broisat, J Toczek, LS Dumas, M Ahmadi, S Bacot, P Perret, L Slimani, ... Journal of Nuclear Medicine 55 (10), 1678-1684 , 2014 2014 Citations: 61
Matrix metalloproteinase inhibitor, doxycycline and progression of calcific aortic valve disease in hyperlipidemic mice JJ Jung, M Razavian, HY Kim, Y Ye, R Golestani, J Toczek, J Zhang, ... Scientific Reports 6 (1), 32659 , 2016 2016 Citations: 45
Synthesis and in vitro and in vivo evaluation of MMP-12 selective optical probes T Bordenave, M Helle, F Beau, D Georgiadis, L Tepshi, M Bernes, Y Ye, ... Bioconjugate chemistry 27 (10), 2407-2417 , 2016 2016 Citations: 43
Matrix metalloproteinase–targeted imaging of lung inflammation and remodeling R Golestani, M Razavian, Y Ye, J Zhang, JJ Jung, J Toczek, K Gona, ... Journal of Nuclear Medicine 58 (1), 138-143 , 2017 2017 Citations: 39
Homeostatic, non-canonical role of macrophage elastase in vascular integrity M Salarian, M Ghim, J Toczek, J Han, D Weiss, B Spronck, ... Circulation research 132 (4), 432-448 , 2023 2023 Citations: 34
Preclinical evaluation of RYM1, a matrix metalloproteinase–targeted tracer for imaging aneurysm J Toczek, Y Ye, K Gona, HY Kim, J Han, M Razavian, R Golestani, ... Journal of Nuclear Medicine 58 (8), 1318-1323 , 2017 2017 Citations: 31
Optical imaging of MMP-12 active form in inflammation and aneurysm M Razavian, T Bordenave, D Georgiadis, F Beau, J Zhang, R Golestani, ... Scientific reports 6 (1), 38345 , 2016 2016 Citations: 31
Hydroxamate-based selective macrophage elastase (MMP-12) inhibitors and radiotracers for molecular imaging K Gona, J Toczek, Y Ye, N Sanzida, A Golbazi, P Boodagh, M Salarian, ... Journal of medicinal chemistry 63 (23), 15037-15049 , 2020 2020 Citations: 25
FDG PET imaging of vascular inflammation in post-traumatic stress disorder: A pilot case–control study J Toczek, AT Hillmer, J Han, C Liu, D Peters, H Emami, J Wu, I Esterlis, ... Journal of Nuclear Cardiology 28 (2), 688-694 , 2021 2021 Citations: 23
Elucidating atherosclerotic vulnerable plaque rupture by modeling cross substitution of ApoE−/− mouse and human plaque components stiffnesses J Ohayon, N Mesnier, A Broisat, J Toczek, L Riou, P Tracqui Biomechanics and modeling in mechanobiology 11 (6), 801-813 , 2012 2012 Citations: 22
Computed tomography imaging of macrophage phagocytic activity in abdominal aortic aneurysm J Toczek, P Boodagh, N Sanzida, M Ghim, M Salarian, K Gona, G Kukreja, ... Theranostics 11 (12), 5876 , 2021 2021 Citations: 16
Novel matrix metalloproteinase 12 selective radiotracers for vascular molecular imaging J Toczek, T Bordenave, K Gona, HY Kim, F Beau, D Georgiadis, I Correia, ... Journal of medicinal chemistry 62 (21), 9743-9752 , 2019 2019 Citations: 16
Assessing Low Levels of Mechanical Stress in Aortic Atherosclerotic Lesions From Apolipoprotein E −/− Mice—Brief Report A Broisat, J Toczek, N Mesnier, P Tracqui, C Ghezzi, J Ohayon, LM Riou Arteriosclerosis, thrombosis, and vascular biology 31 (5), 1007-1010 , 2011 2011 Citations: 15
