MicroRNA as a Biological Drug and Recovery of Myocardial Infarction Exosome Jianyi (Jay) Zhang, MD, PhD Professor of Medicine, of Engineering University of Alabama - Birmingham UAB 1
Rebuilding the Failing Heart with Cell Therapy Road Blocks to Overcome 1. Low engraftment rate 2. Increased arrhythmic potential 3. Potential Mechanisms of Actions 2
Roadblock 1: Low engraftment rate Strategies: hipsc - HLA I/II KO to generate Universal Cell Lines In collaboration with Townes lab at UAB Local Delivery : Myocardial Tissue Equivalent Patch using hipsc- tri lineage Cardiac Cells 3
Roadblocks 2: Arrhythmias: Microenvironment of graft and recipient heart Strategies: Deciphering the mechanisms of arrhythmias Ectopic center or reentry? Ca2+ and Action potential propagation passing the interface: Optical mapping and micro impedance hipsc Gap Junction Protein Over Expression:Cx43 4
Roadblock 3 Unknown Potential Mechanisms of Actions Strategy 3: Local Delivery : Myocardial Tissue Equivalent Patch using hipsc- tri lineage Cardiac Cells The potential mechanisms of actions from the perspective of the regulations in myocardial perfusion, metabolism and function in the in vivo heart 5
hipsc-cm TE graft 4 weeks after transplantation (A) (C) Interface Patch Host (D) Interface (F) Interface Patch HNA DAPI Patch ctnt Ki67 PCs DAPI Wendel J et al 2015 SCTM
Significant Angiogenesis Support the CM Grafts (week 4) (A) (B) Patch Patch IB4 DAPI (C) Interface H &E Wendel J et al
Fabrication human cardiac muscle patch CM cluster Fabrication of hipsc-cm Patch Zhang L Circ 2014
hipsc Derived tri- Lineage Cardiovascular Cells for Postinfarction LV Remodeling Fabrication of Larger and Thicker Human Myocardial Tissue Equivalent UAB
Fabrication of Larger and Ticker Myocardial Tissue Equivalent (MTE) Figure 2. Left, Fibrin hcmp patch (2 cm x 4 cm) containing 10 million hipsc-cms, 5 million hipsc-ecs, and 5 million hipsc-smcs. After 7 days in culure, the hcmp beats regularly at rate of 100 beat/min. Right, Two rectangle fibrin hcmp were sutured on the surface of a pig heart that exposed to 60 minutes of no flow ischemia reperfusion
Electrical pacing of human cardiac MTE patch
Optical mapping of V m in cardiac patch Conduction velocity = 15.1 cm/s APD 50 = 306 ms APD 80 = 353 ms
APD (ms) CV (cm/s) Rate dependence of APD and conduction velocity 400 17 380 16 360 340 320 300 280 APD50 APD80 15 14 13 12 11 10 260 9 240 500 700 900 1100 1300 8 500 700 900 1100 1300 CL (ms) CL (ms)
RH237 GCaMP6 Activation Time (ms) Dual mapping of ventricle and implanted patch. A 10 ms 20 ms 30 ms 5 mm 40 ms 50 ms 60 ms B C Ventricle CV = 60.5 cm/s 5 mm 30 25 20 15 10 5 0 Patch CV = 29.2 cm/s
CM Maturation in vivo hctnt DAPI Merged Day 1 hctnt DAPI Merged Day 28
Completely Noninvasive Cardiac MR Spectroscopy at 7T/65CM Magnet
Patching the Heart: Myocardial Repair from Within or Outside Working hypothesis MSCs hesc/ips -VCs hipsc- VCs + CMs Infarct size reduction DE MRI TUNEL ctnt BrdU CPC Perfusion and Chamber function Perfusion Cine CD31 SMA Metabolism UAB
Acknowledgements: Zhang lab Collaborators : Tranquillo, Garry, UMN Kamp, Ge, UW-Madison Townes, Rogers, Fast, Walcott; UAB Bursac, Duke NIH Grants : NIH RO1s HL67828, HL 95077, HL114120, UO1 HL100407 21
Acknowledgements: Zhang lab Ø Cardiac Repair using Stem Cells Ø Myocardial energetics 31 P MR spectroscopy Collaborators : Tranquillo, Garry, UMN Kamp, Ge, UW-Madison Townes, Rogers, Fast; UAB NIH Grants : NIH RO1s HL67828, HL 95077, HL114120, 22 UO1 HL100407
A Non-human Primates Study Hypoxia Preconditioning Cardiac Function Evaluation N-MSCs NHP Model (N=49) Intramyocardial Injection HP-MSCs Cell Tracing Cell Survival PET IH CD4 CD8 Cardiac Metabolism Electromechanical Stability (PES) Telemetry Immune Modulation Molecular Mechanism Arrhythmia detection Circres JAN 2016
Potential Mechanisms of Actions Strategy 3: Local Delivery : Myocardial Tissue Equivalent Patch using hipsc- tri lineage Cardiac Cells 24
Arteriole density (mm -2 ) Resistance vessel density (week-4) CD31 SMA ctnt MI Patch 500 400 300 200 100 0 p<0.05 p<0.05 MI Patch Cell P+Cell
BZ myocardial energetics In vivo 31 P MR spectroscopy * * # Unidirectional ATP utilization rate: ATP ADP + Pi * * # Xiong Q et al Circulation. 2013
BZ myocardial Wall stress, Flux ATP Pi contractile function Normal MI CELL -BZ -BZ -BZ -BZ Laplace law: wall stress (P) LVSP radius 2 thickness
Over stretched myocytes in failing hearts Murakami Y et al Circ 1999
31 P spectra were acquired with a 3D ultra-short TE chemical shift imaging (UTE-CSI) sequence in a normal adult mongrel dog acquired on a Magnetom 7T scanner 29
Peak Force Generated (mn) Force (mn) Fabrication of a Myocardial Tissue Equivalent Disaggregated neonatal rat cardiac cells seeded into fibrin gel 7 days 7 days F-actin ctni DAPI 3.5 Twitch Force Generation 9.5 9 8.5 1 H z 9.5 9 8.5 2 H z 3 8 7.5 8 7.5 2.5 7 7 2 6.5 6 6.5 6 CX43 ctnt DAPI 1.5 1 0.5 0 1 2 3 4 Stimulation Frequency (Hz) 5.5 0 500 1000 1500 2000 2500 3000 3500 4000 4500 5000 7.4 7.2 7 6.8 6.6 6.4 6.2 6 3 H z 5.5 0 1000 2000 3000 4000 5000 6000 9 8.8 8.6 8.4 8.2 8 7.8 7.6 4 H z 0 1000 2000 3000 4000 5000 6000 7.4 0 1000 2000 3000 4000 5000 6000 Time (ms)
Myocardial Tissue Equivalent Patch Study Groups: 1) Sham, (n=5) 2) MI, (n=6) : Ligation Only 3) TE CM-, (n=5): MI+ tissue equivalent constructed without CMs 4) TE CM+, (n=7): MI + tissue equivalent containing CMs Week 1 and 4 follow up with ECHO Wendel J TE 2014
Percent of LV anterior wall Engraftment of Tissue Equivalent to the Host Myocardium noncm Host Infarct Size 100 80 ctnt F-actin DAPI Host Myocardium Patch noncm 20um Sham 60 40 * 20 0 CM patch * noncm patch MI only CM Host Interface Graft F-actin Host Myocardium Patch CM 100um (D) 50um
Center for Magnet Resonance Research 9.4T-65cm magnet 7T-90cm magnet Aa b c d e f
[ADP] K x [(PCr) /(ATP)] -1 k [PCr] [ADP] [ATP] [Cr]
Proteins downregulated in MI Proteins up-regulated in MI Normal MI MI+iPSC -VC Myocardial Differential Protein Expression Profile Changes in response to Cell Patch Therapy AA C -1.0 0.0 1.0 Regulation of metabolic process B Cytoskeleton organization Regulation of cell morphogenesis Fig. 6. Number of proteins identified from heart tissue Electron transport chain homogenate using gene ontology annotation ATP Synthesis coupled for cell electron transport compartment analysis.
Recipient myocardial protein expression profile changes
Identify the grafted hipsc-cms (week-4) GFP DAPI ctnt DAPI Merged Phase contrast
Cell transplantation reduced apoptosis (day-3) TUNEL + ctni ctni DAPI Merged MI Patch only P + Cell
c-kit + CV PC density (cm -2 ) Cell transplantation activated c-kit + CV progenitor cell (week-4) c-kit DAPI 1500 1200 * MI c-kit ctnt DAPI Patch Cell 900 600 300 MI Patch Cell 0 MI Patch Cell
Summary The preliminary results suggest the capacity of a fibrinbased cardiac tissue equivalent to engraft 4 weeks after transplantation, which is accompanied by a reduction of infarct size, and improvement of LV chamber function. The mechanisms of the reduced infarct size are not clear, but are likely related to the cytokine related protective effect. The optimized synergetic effects of the cytokine signaling pathways are depend upon communications between myocytes and non-cm cardiac cells.
Identify the grafted hipsc-smcs (week-4) GFP DAPI SMA DAPI GFP SMA ctni DAPI Bright field
Identify the grafted hipsc-ecs (week-4) hcd31 DAPI GFP ctnt DAPI hcd31 ctni DAPI Bright field 42
Angiogeic profile of EC and SMC conditioned medium EC SMC CM Growth factor, cytokine Angiogenin 58136 14962.3 56303.5 Angiopoietin-1 1273 8030.25 3967.75 Angiopoietin-2 12206.3 790 1060 IL-6 46934.3 2234.5 5450.5 PDGF-BB 2140.25 306 25.75 VEGF 25.5 18 513.5 Chemokine TGF-beta1 654.75 758.25 706.5 Growth regulated protein 57706.5 3516.25 8524.25 IL-8 23679.8 2910.5 4585 MCP-1 65447 65447 65442.8 RANTES 1539 1510 96 MCP-3 1200.25 524.5 16919 Inhibitor of metalloproteinases TIMP-1 13565.8 18635.5 13430.5 TIMP-2 7394.5 12428.3 8581 Angiogeic inhibitor Angiostatin 219 244.25 267.25 Endostatin 575 159.75 310.75 Protease MMP-9 731 196.75 163.25 Plasminogen activator u PAR 3236.5 2618.5 1101 Angiogeic receptor VEGF R2 700.75 150 185.5 VEGF R3 299.5 326.5 299.5
Aims To develop an efficient hipsc-cm selection protocol To examine the efficiency of a patch and microspheres based enhanced delivery of hipsc - 3 lineage cardiovascular cells for myocardial repair using an immuno-suppressed porcine model of postinfarction LV remodeling: - engraftment rate, - vascular density and myocardial perfusion, - myocardial protection and apoptosis - tracking the endogenous CV PCs with BrdU Using novel NMR technology to examine the myocardial bioenergetics and ATP turnover rate in the in vivo hearts with or without cell transplantation The electrophysiology stability was examined by loop recorder and recipient myocardial differential protein expression profile by proteomics
3D Porous PEGylated Fibrin patch for enhanced delivery of hipsc 3-lineage cardiac cells PEG GM Zhang, G Tissue Engineering 2007
Gelatin microsphere for IGF delivery
Percentage Percentage Cell engraftment rate (week-4) 15% 50% 10% 8.97±1.8% 40% 30% 26.76% 33.44% 39.80% 5% 20% 10% 0% Total cell engraftment rate 0% hipsc-cms hipsc-ecs hipsc-smcs Quantitative PCR (qpcr) for human Y chromosome Dual immunostaining