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Cardiac MRI of Magnetically-Labeled Annexin Detects Cell Injury, In Vivo Rajesh Dash 1,2,3 , Jaehoon Chung 1 , Trevor Chan 1 , Mayumi Yamada 1 , Joëlle Barral 1 , Dwight Nishimura 1 , Paul Simpson 2,3 , and Phillip Yang 1 Stanford University 1 ; San Francisco VAMC 2 ; and UCSF Medical Center 3 BACKGROUND • Drug-induced, ischemic, and non-ischemic cardiomyopathies are associated with marked cardiac apoptosis • Early , non-invasive detection of cardiac apoptosis or cell injury may improve patient outcomes through targeted medical and cell- based therapies • Annexin V (ANX), which binds externalized membrane-bound phosphatidylserine, can detect early apoptosis • Cardiac MRI exhibits high spatial and temporal resolution and can detect magnetically tagged proteins and cells, in vivo HYPOTHESES • Can ANX, which was previously conjugated to superparamagnetic iron oxide (SPIO), detect cardiac apoptosis/cell injury, in vivo , via T2-MRI? • Is the in vivo pattern of ANX-SPIO T2* signal loss distinct in oxidative (Doxorubicin) vs ischemic cardiac injury ? METHODS Generation of ANX-SPIO Conjugate Protein Purified human ANX V was previously generated using purified human ANXV protein and Feridex iron oxide (11.2mg Fe/ml soln), through a series of oxidation/ reduction steps. In Vitro MRI Detection of Apoptosis Using ANX-SPIO T2-MRI of ANX-SPIO was previously found to identify small populations of apoptotic cardiomyocytes (CMs), fibroblasts (FBs), and mesenchymal stem cells (mMSCs) in culture, with high specificity and sensitivity. In Vivo Detection of Apoptosis Using ANX-SPIO 20μg of ANX-SPIO was delivered via tail vein into FVB/n mice 48 hours after myocardial infarction (MI) surgery, and 48 hours after intraperitoneal Doxorubicin (DOX, 25mg/kg) injection. Previous studies of this dose of DOX showed extensive cardiac fibrosis, cell death, and ventricular dysfunction at 1 week. Cardiac MRI evaluated T2* signal loss within the myocardium at days 2, 7, and 28 post-MI. At day 28, MRI’s were performed before and after re-injection of ANX-SPIO. Similarly, Cardiac MRI was performed on DOX-treated animals on days 2 and 7. A 3 Tesla, GE MRI system was used, employing cardiac and respiratory gating, with a custom surface loop receiver coil, designed by Dr. Barral and Dr. Nishimura. Qualitative differences in T2* signal loss patterns were assessed. All animal work was in compliance with APLAC safety and ethical requirements, APLAC Protocol #17946. Figure 1: Expression & Purification of Annexin V (ANX) Protein CONCLUSIONS • ANX-SPIO detects areas of cardiac injury, in vivo • The pattern of T2* signal loss created by ANX-SPIO is distinct in DOX-treated versus MI-mouse hearts • Future studies: • Histopathological confirmation of iron stain, caspase activity • Assessment of dynamic changes following therapy ACKNOWLEDGEMENTS This work was made possible through an AHA Western States Affiliates Post-Doctoral Research Fellowship (R.D.), and from R01 (P.S), and K series (P.Y.) grants from the NIH. Special thanks to Grant Hoyt for his surgical expertise. No financial disclosures or conflicts of interest to report. 2 kb 1.5 kb 600 bp 964b p +C -C 1 2 3 p 1-p dNTP Colony Controls GST T7 LacI Amp R ANX-V 964 BP 100 75 50 kDa 63 kDa 1 2 3 Colony Protein Purification Fig 1: Expression / Purification of ANXV (A) GST-ANXV plasmid construct with T7 bacterial promoter & AmpR gene. 964bp GST-ANXV fragment. (B) PCR using GST-ANXV primers. Colonies 1,2, and 3 show bands at expected fragment size. Negative controls using only primers (p), colony 1 without primers (1-p), & dNTPs alone are also shown. (C) Coomassie Blue staining of SDS-PAGE gel from Glutathione column elutions. 63kDa band is the expected fusion protein product. A) C) B) Figure 2. T2-MRI Signal Linearly Correlates with Apoptosis R 2 = 0.8872 p<0.05 by F-test 0 50 100 150 200 250 300 0 10 20 30 40 50 FITC+ / PI - (apoptotic) cells B) A) T2 Signal (Arbitrary Units) DOX 10 min DOX 60 min Figure 2: A) Increased signal with increased doxorubicin (DOX) exposure time. Left: T2-weighted MRI GRE images of mouse mesenchymal stem cells cultured on 35mm plates, treated with 1μM DOX for 10 or 60 minutes, followed by ANX-SPIO incubation. Right: Corresponding ANX-FLUOS (green) and propidium iodide (red) fluorescent signal for each DOX group. B) Linear correlation between degree of T2-weighted MRI signal and # of FITC+/PI- cells for similarly treated groups in culture. Figure 3. Specific ANX-SPIO Interaction - Competition Binding 0 10 -2 10 -1 1 10 10 -3 100 Log [ANX-SPIO] (1 = 2ug protein) # of FITC+ cells/well K i = 0.2uM N=3 Figure 3. A) MRI & Fluorescent images of mMSCs stained with ANX-SPIO +/- ANX- FLUOS post-DOX. B) Competition curve using Rat Neonatal CMs post- DOX, incubated with a saturating dose of ANX- FLUOS + increasing doses of ANX-SPIO. Plots were curve-fit using a non-linear regression to obtain K i (n=3, p<0.05). Control + Anx-SPIO OR ANX-Fluos DOX + Anx-SPIO + Anx-Fluos DOX + Anx-SPIO OR Anx-Fluos B) A) Figure 6. ANX-SPIO & MRI Detect In Vivo Cell Injury After DOX Figure 6. 20ug ANX-SPIO delivered via tail vein injection, 48hrs after DOX injection (25mg/ kg i.p.). MRI images were obtained with cardiac and respiratory gating. GRE images of 4 chamber views from DOX + SPIO alone (left) versus DOX+ ANX-SPIO (right) mice are shown. Note the speckled T2* signal loss that is diffusely distributed in the DOX+ANX-SPIO mouse heart. Figure 4. MRI-Detectable, Differential DOX Sensitivity Between Cell Types Figure 4: MRI-detectable DOX-susceptibility differences between Rat Neonatal CMs, Mouse Mesenchymal Stem Cells, and Rat Neonatal Cardiac Fibroblasts. Time course of apoptotic signal from FLUOS-ANXV (blue) and T2-weighted MRI signal (yellow) over increasing DOX exposure times. (*p<0.05 versus control, # p<0.05 versus 30 min DOX timepoint; ANOVA) HOURS of DOX Exposure (1μM) 0 0 0.5 1 2 3 24 50 100 * * Rat Neonatal CM * * Rat Cardiac Fibroblasts Mouse Mesenchymal SC # # % Max T2* Signa l Loss • - p<0.05 vs Control (CM) # - p<0.05 vs Control (mMSC) - p<0.05 vs Control (FB) N= 4 Figure 5: Protocols for DOX and MI Imaging MRI 24-48 hours DOX 25mg/kg i.p. ANX-SPIO Infusion MRI 48 hours LAD Ligation ANX-SPIO Infusion 5 days or 4 weeks MRI ANX-SPIO Infusion MRI @ 48hr post-DOX (25mg/kg i.p.) DOX + SPIO alone DOX + ANX-SPIO Long-Axis View: LV LV RV IVS RV IVS LV LV RV Short-Axis View: IVS Figure 7. ANX-SPIO & MRI Detect Cell Injury In Vivo After MI Figure 7. Acute and Chronic GRE images of exhibiting T2* signal loss in the anteroapex following LAD ligation. The transmural, perinfarct pattern is in contrast to the speckled pattern of DOX treated hearts. AW IW LV LA MI - Acute Phase LW LV IVS RV Chronic MI 4 weeks later 3 Tesla: GRE TR100/TE15/FA60/FOV4/ST1mm 3T GRE TR 100/TE 15/FA 60/FOV 4/ST 0.8mm In Vitro Validation In Vivo Application
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