This laboratory develops and explores Magnetic Resonance (MR) visible probes to tag intracellular gene transcription activator proteins and gene transcripts for non-invasive MR imaging. The rationale for such development is that molecular biological assays have been advanced our understanding of human diseases, but they are not performed in human brains except in late tumor cases. With recent translation of stem cell therapy in neurological degenerative diseases and bone marrow regenerative cells can be recruited to repair brain damage, biopsy sampling defeats the benefits of stem cell therapy.
Three animal models are used to validate our development before applications in humans.
1. Angiogenesis during Brain Repair: Approximately 250,000 Americans die each year as a result of cardiac arrest. Fewer than 10% of those who suffer cardiac arrest survive more than 6 months, and those fortunate to survive can develop residual neurological deficits. Several mesenchymal- and hematopoietic-lineage cells have been used to validate localized angiogenesis; these cell types include neuronal, glial, and endothelial biomarkers such as proteins RNA and DNA (Fig 1). However, such regenerative cells have been detected by several unique antibodies in biopsy or postmortem samples, which do not benefit regeneration purpose of pericytes at all. The challenge is to bring these top notch molecular assays into in vivo conditions for future clinical application.
Currently, MRI and positron emission tomography are the only non-invasive tools available for identifying the location of damaged brain tissue and the important process of tissue repair including therapy using stem cells and the formation of cerebral neo-vascularization (angiogenesis). We have developed a nanotechnology to detect molecular events in living brains. We now demonstrated a noninvasive technique to detect Nestin mRNA in pericytes, by delivering dual targeting agents to the same mice (Fig 2). We detected a repair patch of new blood vessels after cerebral ischemia in mice, achieving a resolution of 0.06 mm3 using MR contrast agent of 5-30 nm (dia) to identify angiogenesis. Such multipotent regenerative cells can be differentiated as new pericytes in microvessels, or as muscle cells in larger vessels.
The bases of nanotechnology on gene tagging in living rodent brains for mechanistic research imaging are illustrated in Fig 3. There are three unique components in specially made MR contrast agent (panel A): a carrier (phosphorothioate-modified micro DNA, or sODN) for endocytosis (Beltinger et al., 1995), a DNA sequence for tagging mRNA of interest by hybridization in specific cells and a contrast agent cargo (MR-visible contrast agents) for MRI. These are unconventional and exceptionally innovative by application: (1) to utilize a short DNA to carry large SPION (~30 nm in diameter) into the cell in living brains, (2) a stable NA and biotin linkage to survive in body fluids. (3) to validate specific cell tagging by TEM, and (4) to compare sensitivity with RT-PCR or TaqMan analysis, (5) to image mRNA activities with an optimal concentration of MR contrast agent within the confinement of a cell for MRI signal using high field strength (9.4Tesla) MR system with (6) a low dose of the sODN dose (3 pmol per mouse) effecting specific binding according to Watson and Crick base-pairing (7) without a measurable gene knockdown effect, and (8) is cleared out from the cell within few hours so that (8) longitudinal and repeating MRI is possible. This nanotechnology has future application in clinical trial for live brains to identify gene action and specialty cell mapping.
Lastly, but not the least, we have begun testing a range of low to high doses (40-1000 mg per kg, icv) to NHP and did not produce lethal effect in the rhesus macaques, except high dose may reduce clearance with 24 hrs (Fig 4). This is the first step toward clinical applications.
2. Effect of Amphetamine abuse: Drug addiction is a major health problem that severely hampers the productivity of many members of our society. While studies indicate that drug addiction results from combined influences of genes and environment, recent studies suggest that epigenetic modifications of gene transcription factors may play an important role in the development of addictions in humans. We have gene transcript target MRI contrast agents to intracellular mRNA of cFos, FosB, deltFosB, GFAP and HDAC5 proteins and microRNAs of non-coding RNA transcripts, such as miR9, miR2861. Currently, we are developing intracellular protein-targeting MRI contrast agents.
3. Retinopathy: Retinopathy is one of many causes for blindness due to ischemia and diabetes. Patients with diabetes are at risk of proliferative diabetic retinopathy, involving capillary degeneration and loss of pericytes (mural cells). The ability to image and quantitatively measure pericytes in vivo in inaccessible retinal tissue would both enhance our understanding of diabetic retinal degeneration and our ability to develop and evaluate therapies. We have developed a pericyte-specific contrast agent using phosphorothioate-modified antisense DNA (sODN) for in vivo reporting and quantitative characterization of pericytes. When labeled with superparamagnetic iron oxide nanoparticles (SPION), these sODN can be used for multimodal detection by magnetic resonance imaging (MRI) in vivo and electronic microscopy (EM) ex vivo. We have designed several SPION-sODNs that co-register cohorts of cells with a resolution of < 0.03 mm3. Administered by intraperitoneal (i.p.) injection or eye drop, these SPION-sODNs can detect expression of cerebral matrix metalloproteinase 9 (MMP9), as well as Actin and Nestin mRNA over a course of 14 to 17 weeks in a C57black6 mouse model of forebrain ischemia that involves leakage of the blood-brain barrier (BBB) and blood-retina barrier (BRB). The specificity of this contrast agent is mediated by intracellular retention of sODN that binds to the intracellular mRNA target and the exclusion of free or non-targeting sODN. This is true of all living cells including multipotent PC12 cells, cells in fresh brain slices and living mouse brains. Given that the eye exhibits proliferating BRB leakage as diabetes progresses, we propose to apply this method to detect retinal pericytes.
Progress Report (2011 - present):
Christina H. Liu, Jia Q Ren, Zerong You,Jinsheng Yang, Charngming Liu, Ratika Uppal, and Philip K. Liu. Neural Progenitor Identification in Living Brains by Dual Gene Transcript-Targeted MRI; the FASEB Journal, 11/27/2011.
Liu, Philip K, and Christina H. Liu. Gene Targeting MRI: Nucleic Acid-Based Imaging and Applications (Ch. 18). (Michel Modo & Jeff W.M. Bulte, eds), Springer Science+Business Media. 2011
Shizuka Minamishima, Kotaro Kida, Patrick Y. Sips, Huifang Wang, Kentaro Tokuda, Shizuko Kosug, Joseph B. Mandeville, Emmanuel S. Buys, Peter Brouckaert, Philip K. Liu, Kenneth D. Bloch, Fumito Ichinose. Inhaled Nitric Oxide Improves Long-term Outcome After Successful Cardiopulmonary Resuscitation in Mice. Circulation, 124(15), 1645-53, 2011. PMCID 3199136.
Renhua Wu, Charng-Ming Liu, Philip K Liu, and Phillip Zhe Sun. Improved measurement of labile proton concentration-weighted chemical exchange rate (kws) with experimental factor-compensated and T1-normalized quantitative chemical exchange saturation transfer (CEST) MRI, Contrast Media and Molecular Imaging, accepted Dec, 27. 2011.
Christina H. Liu, Jinsheng Yang, Jia Q Ren, Charngming Liu, Zerong You,and Philip K. Liu. MRI Reveals Differential Effects of Amphetamine Exposure on Neuroglia in vivo. FASEB J, 27:712-24, 2013.
Liu, CH., Ren, JQ., Liu, CM., Yang, JS, and Liu, PK., Amphetamine-induced Restless Behavior is mediated by Reduction of Monoamine Oxidase-A Activities in the Ventral Tegmental Area of the Mesolimbic Pathway. FASEB J, accepted with revision, 2013, In this study we show that amphetamine exposure significantly (p < 0.05, t test) ameliorates MAO levels in the ventral tegmental area of mesolimbic pathway in mice. We developed an AP-1 aptamer (5ECdsAP1) containing an AP-1 transcription factor (TF) binding sequence for recombinant human AP-1 protein, we shown theranostic application to reduce amphetamine-evoke hyper-activities in mice.
Ongoing Research Support
1R01DA029889-01 (PI: PK Liu) - 07/01/2010–06/30/2014
Title: Aptamer Imaging: A Theranostic Strategy for Substance Abuse, (Exceptional, Unconventional Research Enabling Knowledge Acceleration or EUREKA Award). This proposed project aims to tag intracellular activator protein of the live animal brain for non-invasive MRI.
1R01EB013768-01A1 (PI: PK Liu) - 07/01/2011-06/30/2015
Title: DNA-Based MR Probes for Imaging mRNA Transcripts in vivo. We will study and compare mRNA targeted by phosphorothioate-modified DNA- and RNA-based contrast agents to improve the utility of ggene transcript-targeted MRI.
1R01HL01930-01 (PI: F. Ichinose) - 04/06/2010–04/05/2015
Title: Impact of hydrogen sulfide on outcome of cardiac arrest and cardiopulmonary resuscitation. The application examines the role of hydrogen sulfide (H2S) in ameliorating the adverse effects of cardiopulmonary resuscitation (CPR) following cardiac arrest (CA) in a mouse model. Role on Project: Investigator.
1R01-HL110378-01A1 (PI: Ichinose/Bloch) - 07/01/2012 – 06/30/2016
Title: Improving Outcomes in Cardiac Arrest/CPR with Inhaled Nitric Oxide The goal of this proposal is to determine the beneficial effects of inhaled nitric oxide to promote the recovery from heart attack complicated with the brain and heart injury. Role on Project: Investigator.
P30DK057521-14 (PI: J Avruch) - 2/2013-1/2014
Title: The Boston Area Diabetes Endocrinology Research Center (BADERC) Parent (Pilot and Feasibility Study for MRI of Pericyte to Quantitate Diabetic Retinopathy in vivo (PKLIU)
Completed (most recent four of ten)
NIH R01DA026108-03 (PI: PK Liu) 06/15/2009-05/30/2012
Title: In vivo Profiling of Glial and Neuronal Activities in Psychostimulant Abuse.
NIH R21DA024235 (PI: Philip K Liu) 09/01/2007-08/31/2010
Title: MR Assessment of Altered Gene Expression after Amphetamine Exposure
NIH R21NS057556 (PI: Philip K. Liu) 06/16/2007-05/30/2010
Title: Neurovascular Mechanisms of Brain Function and Diseases
NIH R01NS045845 (PI: Philip K. Liu) 05/01/2003-06/30/2009
Title: Gene Repair in Signal Transduction after CNS Injury
NIH R01NS034810 (PI: Philip K. Liu) 09/01/1996-8/31/2001
Title: Hydroxyl Radical Biology by Cerebral Ischemia