Phillip Harnish

Direct Evaluation of Myocardial Viability and Stem Cell Engraftment Demonstrates Salvage of the Injured Myocardium

RATIONALE The mechanism of functional restoration by stem cell therapy remains poorly understood. Novel manganese-enhanced MRI and bioluminescence reporter gene imaging were applied to follow myocardial viability and cell engraftment, respectively. Human-placenta-derived amniotic mesenchymal stem cells (AMCs) demonstrate unique immunoregulatory and precardiac properties. In this study, the restorative effects of 3 AMC-derived subpopulations were examined in a murine myocardial injury model: (1) unselected AMCs, (2) ckit(+)AMCs, and (3) AMC-derived induced pluripotent stem cells (MiPSCs). OBJECTIVE To determine the differential restorative effects of the AMC-derived subpopulations in the murine myocardial injury model using multimodality imaging. METHODS AND RESULTS SCID (severe combined immunodeficiency) mice underwent left anterior descending artery ligation and were divided into 4 treatment arms: (1) normal saline control (n=14), (2) unselected AMCs (n=10), (3) ckit(+)AMCs (n=13), and (4) MiPSCs (n=11). Cardiac MRI assessed myocardial viability and left ventricular function, whereas bioluminescence imaging assessed stem cell engraftment during a 4-week period. Immunohistological labeling and reverse transcriptase polymerase chain reaction of the explanted myocardium were performed. The unselected AMC and ckit(+)AMC-treated mice demonstrated transient left ventricular functional improvement. However, the MiPSCs exhibited a significantly greater increase in left ventricular function compared with all the other groups during the entire 4-week period. Left ventricular functional improvement correlated with increased myocardial viability and sustained stem cell engraftment. The MiPSC-treated animals lacked any evidence of de novo cardiac differentiation. CONCLUSION The functional restoration seen in MiPSCs was characterized by increased myocardial viability and sustained engraftment without de novo cardiac differentiation, indicating salvage of the injured myocardium.

Dual Manganese-Enhanced and Delayed Gadolinium-Enhanced MRI Detects Myocardial Border Zone Injury in a Pig Ischemia- Reperfusion Model

Background— Gadolinium (Gd)-based delayed-enhancement MRI (DEMRI) identifies nonviable myocardium but is nonspecific and may overestimate nonviable territory. Manganese (Mn2+)-enhanced MRI (MEMRI) denotes specific Mn2+ uptake into viable cardiomyocytes. We performed a dual-contrast myocardial assessment in a porcine ischemia-reperfusion (IR) model to test the hypothesis that combined DEMRI and MEMRI identifies viable infarct border zone (BZ) myocardium in vivo. Methods and Results— Sixty-minute left anterior descending coronary artery IR injury was induced in 13 adult swine. Twenty-one days post-IR, 3-T cardiac MRI was performed. MEMRI was obtained after injection of 0.7 mL/kg Mn2+ contrast agent. DEMRI was then acquired after injection of 0.2 mmol/kg Gd. Left ventricular (LV) mass, infarct, and function were analyzed. Subtraction of MEMRI defect from DEMRI signal identified injured BZ myocardium. Explanted hearts were analyzed by 2,3,5-triphenyltetrazolium chloride stain and tissue electron microscopy to compare infarct, BZ, and remote myocardium. Average LV ejection fraction was reduced (30±7%). MEMRI and DEMRI infarct volumes correlated with 2,3,5-triphenyltetrazolium chloride stain analysis (MEMRI, r=0.78; DEMRI, r=0.75; P<0.004). MEMRI infarct volume percentage was significantly lower than that of DEMRI (14±4% versus 23±4%; P<0.05). BZ MEMRI signal-to-noise ratio (SNR) was intermediate to remote and core infarct SNR (7.5±2.8 versus 13.2±3.4 and 2.9±1.6; P<0.0001), and DEMRI BZ SNR tended to be intermediate to remote and core infarct SNR (8.4±5.4 versus 3.3±0.6 and 14.3±6.6; P>0.05). Tissue electron microscopy analysis exhibited preserved cell structure in BZ cardiomyocytes despite transmural DEMRI enhancement. Conclusions— The dual-contrast MEMRI-DEMRI detects BZ viability within DEMRI infarct zones. This approach may identify injured, at-risk myocardium in ischemic cardiomyopathy.

Manganese-Enhanced Magnetic Resonance Imaging Enables In Vivo Confirmation of Peri-Infarct Restoration Following Stem Cell Therapy in a Porcine Ischemia-Reperfusion Model.

Background The exact mechanism of stem cell therapy in augmenting the function of ischemic cardiomyopathy is unclear. In this study, we hypothesized that increased viability of the peri-infarct region (PIR) produces restorative benefits after stem cell engraftment. A novel multimodality imaging approach simultaneously assessed myocardial viability (manganese-enhanced magnetic resonance imaging [MEMRI]), myocardial scar (delayed gadolinium enhancement MRI), and transplanted stem cell engraftment (positron emission tomography reporter gene) in the injured porcine hearts. Methods and Results Twelve adult swine underwent ischemia–reperfusion injury. Digital subtraction of MEMRI-negative myocardium (intrainfarct region) from delayed gadolinium enhancement MRI–positive myocardium (PIR and intrainfarct region) clearly delineated the PIR in which the MEMRI-positive signal reflected PIR viability. Human amniotic mesenchymal stem cells (hAMSCs) represent a unique population of immunomodulatory mesodermal stem cells that restored the murine PIR. Immediately following hAMSC delivery, MEMRI demonstrated an increased PIR viability signal compared with control. Direct PIR viability remained higher in hAMSC-treated hearts for >6 weeks. Increased PIR viability correlated with improved regional contractility, left ventricular ejection fraction, infarct size, and hAMSC engraftment, as confirmed by immunocytochemistry. Increased MEMRI and positron emission tomography reporter gene signal in the intrainfarct region and the PIR correlated with sustained functional augmentation (global and regional) within the hAMSC group (mean change, left ventricular ejection fraction: hAMSC 85±60%, control 8±10%; P<0.05) and reduced chamber dilatation (left ventricular end-diastole volume increase: hAMSC 24±8%, control 110±30%; P<0.05). Conclusions The positron emission tomography reporter gene signal of hAMSC engraftment correlates with the improved MEMRI signal in the PIR. The increased MEMRI signal represents PIR viability and the restorative potential of the injured heart. This in vivo multimodality imaging platform represents a novel, real-time method of tracking PIR viability and stem cell engraftment while providing a mechanistic explanation of the therapeutic efficacy of cardiovascular stem cells.

Manganese-enhanced MRI detects live human amnion-derived mesenchymal stem cells in vivo after transplantation and restoration of myocardial function in a pig ischemia-reperfusion injury model

Summary Human Amnion-derived Mesenchymal Stem Cells (hAMSCs) were transplanted into the infarct and periinfarct regions of a pig ischemia-reperfusion model. The hAMSC therapy improved cardiac systolic function post-MI, compared to control animals, and Cardiac MRI with Manganese-Enhanced MRI (MEMRI) was able to detect increased CNR from live populations of hAMSCs within infarct and peri-infarct zones, as confirmed by human nuclear antigen (hNA) immunostaining. Background

Dual contrast enhanced cardiac MRI using manganese and gadolinium in patients with severe ischemic cardiomyopathy detects the peri-infarct region (PIR)

Background Delayed Enhanced MRI (DEMRI) with gadolinium (Gd) is used as gold standard for diagnosis of myocardial infarction. However, the non-specific property of Gd overestimates the infarct size. Conversely, manganese (Mn2+) enters only the live, active cardiomyocytes via L-type Ca2+ channels. From our earlier work in animal MI models, manganese-enhanced MRI (MEMRI) has demonstrated its utility in identifying the viable, nonviable, and injured myocardium. We performed the “first in human” dual-contrast MEMRI-DEMRI to assess the efficacy of MEMRI-DEMRI to identify the periinfarct region (PIR) in patients with severe ischemic cardiomyopathy (ICM).

REGENERATIVE CHANGES OF THE PERI-INFARCT INJURY ALLOWS SUSTAINED RESTORATION OF THE INJURED MYOCARDIUM

The human amniotic mesenchymal stem cells (hAMSCs) exhibit immunomodulatory, partially pluripotent, and precardiac properties. They express the c-kit cardiac progenitor marker and are readily reprogrammed to iPSCs (MiPSCs). Dual contrast manganese-enhanced MRI and delayed-enhanced MRI (MEMRI-DEMRI)

DUAL CONTRAST CARDIAC MRI FOR EVALUATION OF TELMISARTAN AND AMLODIPINE COMBINATION THERAPY IN THE DIABETIC MURINE MYOCARDIAL INJURY MODEL

Background Combination therapy has been shown to improve cardiovascular outcomes in hypertensive, diabetic patients. Amlodipine demonstrates beneficial pleiotropic effects including improved endothelial function, enhanced angiogenesis, and reduced cardiovascular risk. Telmisartan exhibits similar beneficial cardiovascular effects in diabetic patients. Amlodipine in combination with telmisartan may provide a synergistic benefit. In this study, dual contrast CMR, using manganese (manganese-enhanced MRI; MEMRI) and gadolinium (delayed-enhanced MRI; DEMRI), will evaluate the viable myocardium and myocardial scar, respectively, to investigate the underlying mechanism of combined telmisartan and amlodipine therapy. Methods Acute myocardial injury (AMI) was created by ligation of the left anterior descending coronary artery (LAD) in db/db mice. The mice were allocated to two groups: (1) tap water (n = 17) and (2) addition of telmisartan (10 mg/kg/day) and amlodipine (10 mg/kg/day) in the tap water per os ad libitum following 1 week of convalescence after AMI (n = 23). Dual contrast CMR with manganese (MEMRI) and gadolinium (DEMRI) was performed weekly with treatment to obtain LVEF and assess viable myocardium and scar myocardium. After the 4-week follow up period, contractile function was assessed by pressure-volume (PV) loop analysis. Flow cytometry of venous blood was performed to determine the percentage of circulating endothelial progenitor cells. Real-time PCR of the explanted myocardium was performed to evaluate the expression of collagen I and III, CTGF, TGFb, fibronectin and Akt. Results We demonstrate significantly improved left ventricular function at weeks 1, 2, and 4 following combined telmisartan and amlodipine therapy (32.0% ± 4.8%*, 31.8% ± 3.9%*, and 35.3% ± 2.3%*) compared to control (18.9% ± 1.5%, 21.8% ± 1.3%, and 15.7% ± 3.4%, *p < .05). PV loop analysis also demonstrates improved systolic and diastolic parameters with significantly increased maximum pressure and dp/dt max and significantly decreased LV end-diastolic pressure and Tau. MEMRI demonstrates significantly increased viable myocardial volumes in the treated mice (87.2% ± 0.92%*, 90.2% ± 1.3%*, and 91.4% ± 1.1%*) compared to control (80.5% ± 7.6%, 79.9% ± 3.1%, and 79.9% ± 3.1%, *p<0.05) throughout the study. DEMRI scar volumes initially show no difference but demonstrate a significantly decreased scar volume after 4 weeks of treatment. Furthermore, the measurement of endothelial progenitor cells demonstrate a dramatic increase in the treated compared to control mice (98.1% ± 0.25%* vs. 5.0% ± 2.6%, *p<0.05). However, no differential expression of fibrotic genes by PCR was observed. Conclusions

Manganese-Enhanced cardiac MRI (MEMRI) tracks long-term in vivo survival and restorative benefit of transplanted human Amnion-Derived Mesenchymal Stem Cells (hAMSC) after porcine ischemia-reperfusion injury

Manganese-Enhanced cardiac MRI (MEMRI) tracks long-term in vivo survival and restorative benefit of transplanted human Amnion-Derived Mesenchymal Stem Cells (hAMSC) after porcine ischemia-reperfusion injury Rajesh Dash, Paul J Kim, Yuka Matsuura, Xiaohu Ge, Fumiaki Ikeno, Jennifer K Lyons, Ngan F Huang, Scott Metzler, Patricia Nguyen, Shahriar Heidary, Marie-Claude Parent, Tomoaki Yamamoto, John Cooke, Pilar Ruiz-Lozano, Robert C Robbins, Joseph C Wu, Michael V McConnell, Alan Yeung, Phillip Harnish, Phillip C Yang

Dual contrast CMR for evaluation of telmisartan and amlodipine combination therapy in the diabetic murine myocardial injury model

Background Combination therapy has been shown to improve cardiovascular outcomes in hypertensive, diabetic patients. Amlodipine demonstrates beneficial pleiotropic effects including improved endothelial function, enhanced angiogenesis, and reduced cardiovascular risk. Telmisartan exhibits similar beneficial cardiovascular effects in diabetic patients. Amlodipine in combination with telmisartan may provide a synergistic benefit. In this study, dual contrast CMR, using manganese (manganese-enhanced MRI; MEMRI) and gadolinium (delayed-enhanced MRI; DEMRI), will evaluate the viable myocardium and myocardial scar, respectively, to investigate the underlying mechanism of combined telmisartan and amlodipine therapy.