Instructor, Cardiovascular Institute
Honors & Awards
K99/R00 Pathway to Independence Award, National Institute of Health (NIH), National Heart, Lung, and Blood Institute (NHLBI) (08/01/2016 - Present)
Scientist Development Grant, American Heart Association (07/01/2016-07/31/2016)
Western States Affiliate Postdoctoral Fellowship, American Heart Association (2015-2016)
PhD, University College London, UK, Cardiovascular Science (2011)
MRes, University of Manchester, UK, Biological Sciences (2008)
BSc, University Technology of Malaysia, Industrial Biology (2006)
Comparison of Non-Coding RNAs in Exosomes and Functional Efficacy of Human Embryonic Stem Cell- Versus Induced Pluripotent Stem Cell-Derived Cardiomyocytes.
Stem cells (Dayton, Ohio)
Both human embryonic stem cell-derived cardiomyocytes (ESC-CMs) and human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) can serve as unlimited cell sources for cardiac regenerative therapy. However, the functional equivalency between human ESC-CMs and iPSC-CMs for cardiac regenerative therapy has not been demonstrated. Here we performed a head-to-head comparison of ESC-CMs and iPSC-CMs in their ability to restore cardiac function in a rat myocardial infarction (MI) model as well as their exosomal secretome.Human ESCs and iPSCs were differentiated into cardiomyocytes using small molecule inhibitors. Fluorescence-activated cell sorting (FACS) analysis confirmed ∼85% and ∼83% of CMs differentiated from ESCs and iPSCs, respectively, were positive for cardiac troponin T. At a single-cell level, both cell types displayed similar calcium handling and electrophysiological properties, with gene expression comparable to the human fetal heart marked by striated sarcomeres. Sub-acute transplantation of ESC-CMs and iPSC-CMs into nude rats post-MI improved cardiac function, which was associated with increased expression of angiogenic genes in vitro following hypoxia. Profiling of exosomal microRNAs (miRs) and long non-coding RNAs (lncRNAs) revealed that both groups contain an identical repertoire of miRs and lncRNAs, including some that are known to be cardioprotective.We demonstrate for the first time that both ESC-CMs and iPSC-CMs can facilitate comparable cardiac repair. This is advantageous because unlike allogeneic ESC-CMs used in therapy, autologous iPSC-CMs could potentially avoid immune rejection when used for cardiac cell transplantation in the future. This article is protected by copyright. All rights reserved.
View details for DOI 10.1002/stem.2669
View details for PubMedID 28710827
iPSC-derived cardiomyocytes reveal abnormal TGF-ß signalling in left ventricular non-compaction cardiomyopathy.
Nature cell biology
2016; 18 (10): 1031-1042
Left ventricular non-compaction (LVNC) is the third most prevalent cardiomyopathy in children and its pathogenesis has been associated with the developmental defect of the embryonic myocardium. We show that patient-specific induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) generated from LVNC patients carrying a mutation in the cardiac transcription factor TBX20 recapitulate a key aspect of the pathological phenotype at the single-cell level and this was associated with perturbed transforming growth factor beta (TGF-β) signalling. LVNC iPSC-CMs have decreased proliferative capacity due to abnormal activation of TGF-β signalling. TBX20 regulates the expression of TGF-β signalling modifiers including one known to be a genetic cause of LVNC, PRDM16, and genome editing of PRDM16 caused proliferation defects in iPSC-CMs. Inhibition of TGF-β signalling and genome correction of the TBX20 mutation were sufficient to reverse the disease phenotype. Our study demonstrates that iPSC-CMs are a useful tool for the exploration of pathological mechanisms underlying poorly understood cardiomyopathies including LVNC.
View details for DOI 10.1038/ncb3411
View details for PubMedID 27642787
Microfluidic Single-Cell Analysis of Transplanted Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes After Acute Myocardial Infarction.
2015; 132 (8): 762-771
Human induced pluripotent stem cells (iPSCs) are attractive candidates for therapeutic use, with the potential to replace deficient cells and to improve functional recovery in injury or disease settings. Here, we test the hypothesis that human iPSC-derived cardiomyocytes (iPSC-CMs) can secrete cytokines as a molecular basis to attenuate adverse cardiac remodeling after myocardial infarction.Human iPSCs were generated from skin fibroblasts and differentiated in vitro with a small molecule-based protocol. Troponin(+) iPSC-CMs were confirmed by immunohistochemistry, quantitative polymerase chain reaction, fluorescence-activated cell sorting, and electrophysiological measurements. Afterward, 2×10(6) iPSC-CMs derived from a cell line transduced with a vector expressing firefly luciferase and green fluorescent protein were transplanted into adult NOD/SCID mice with acute left anterior descending artery ligation. Control animals received PBS injection. Bioluminescence imaging showed limited engraftment on transplantation into ischemic myocardium. However, magnetic resonance imaging of animals transplanted with iPSC-CMs showed significant functional improvement and attenuated cardiac remodeling compared with PBS-treated control animals. To understand the underlying molecular mechanism, microfluidic single-cell profiling of harvested iPSC-CMs, laser capture microdissection of host myocardium, and in vitro ischemia stimulation were used to demonstrate that the iPSC-CMs could release significant levels of proangiogenic and antiapoptotic factors in the ischemic microenvironment.Transplantation of human iPSC-CMs into an acute mouse myocardial infarction model can improve left ventricular function and attenuate cardiac remodeling. Because of limited engraftment, most of the effects are possibly explained by paracrine activity of these cells.
View details for DOI 10.1161/CIRCULATIONAHA.114.015231
View details for PubMedID 26304668
- HIF-1 reduces ischaemia-reperfusion injury in the heart by targeting the mitochondrial permeability transition pore CARDIOVASCULAR RESEARCH 2014; 104 (1): 24-36
Cross talk of combined gene and cell therapy in ischemic heart disease: role of exosomal microRNA transfer.
2014; 130 (11): S60-9
Despite the promise shown by stem cells for restoration of cardiac function after myocardial infarction, the poor survival of transplanted cells has been a major issue. Hypoxia-inducible factor-1 (HIF1) is a transcription factor that mediates adaptive responses to ischemia. Here, we hypothesize that codelivery of cardiac progenitor cells (CPCs) with a nonviral minicircle plasmid carrying HIF1 (MC-HIF1) into the ischemic myocardium can improve the survival of transplanted CPCs.After myocardial infarction, CPCs were codelivered intramyocardially into adult NOD/SCID mice with saline, MC-green fluorescent protein, or MC-HIF1 versus MC-HIF1 alone (n=10 per group). Bioluminescence imaging demonstrated better survival when CPCs were codelivered with MC-HIF1. Importantly, echocardiography showed mice injected with CPCs+MC-HIF1 had the highest ejection fraction 6 weeks after myocardial infarction (57.1±2.6%; P=0.002) followed by MC-HIF1 alone (48.5±2.6%; P=0.04), with no significant protection for CPCs+MC-green fluorescent protein (44.8±3.3%; P=NS) when compared with saline control (38.7±3.2%). In vitro mechanistic studies confirmed that cardiac endothelial cells produced exosomes that were actively internalized by recipient CPCs. Exosomes purified from endothelial cells overexpressing HIF1 had higher contents of miR-126 and miR-210. These microRNAs activated prosurvival kinases and induced a glycolytic switch in recipient CPCs, giving them increased tolerance when subjected to in vitro hypoxic stress. Inhibiting both of these miRs blocked the protective effects of the exosomes.In summary, HIF1 can be used to modulate the host microenvironment for improving survival of transplanted cells. The exosomal transfer of miRs from host cells to transplanted cells represents a unique mechanism that can be potentially targeted for improving survival of transplanted cells.
View details for DOI 10.1161/CIRCULATIONAHA.113.007917
View details for PubMedID 25200057
miR-21 is associated with fibrosis and right ventricular failure.
2017; 2 (9)
Combined pulmonary insufficiency (PI) and stenosis (PS) is a common long-term sequela after repair of many forms of congenital heart disease, causing progressive right ventricular (RV) dilation and failure. Little is known of the mechanisms underlying this combination of preload and afterload stressors. We developed a murine model of PI and PS (PI+PS) to identify clinically relevant pathways and biomarkers of disease progression. Diastolic dysfunction was induced (restrictive RV filling, elevated RV end-diastolic pressures) at 1 month after generation of PI+PS and progressed to systolic dysfunction (decreased RV shortening) by 3 months. RV fibrosis progressed from 1 month (4.4% ± 0.4%) to 3 months (9.2% ± 1%), along with TGF-β signaling and tissue expression of profibrotic miR-21. Although plasma miR-21 was upregulated with diastolic dysfunction, it was downregulated with the onset of systolic dysfunction), correlating with RV fibrosis. Plasma miR-21 in children with PI+PS followed a similar pattern. A model of combined RV volume and pressure overload recapitulates the evolution of RV failure unique to patients with prior RV outflow tract surgery. This progression was characterized by enhanced TGF-β and miR-21 signaling. miR-21 may serve as a plasma biomarker of RV failure, with decreased expression heralding the need for valve replacement.
View details for DOI 10.1172/jci.insight.91625
View details for PubMedID 28469078
Mitochondrial-Shaping Proteins in Cardiac Health and Disease - the Long and the Short of It!
Cardiovascular drugs and therapy
Mitochondrial health is critically dependent on the ability of mitochondria to undergo changes in mitochondrial morphology, a process which is regulated by mitochondrial shaping proteins. Mitochondria undergo fission to generate fragmented discrete organelles, a process which is mediated by the mitochondrial fission proteins (Drp1, hFIS1, Mff and MiD49/51), and is required for cell division, and to remove damaged mitochondria by mitophagy. Mitochondria undergo fusion to form elongated interconnected networks, a process which is orchestrated by the mitochondrial fusion proteins (Mfn1, Mfn2 and OPA1), and which enables the replenishment of damaged mitochondrial DNA. In the adult heart, mitochondria are relatively static, are constrained in their movement, and are characteristically arranged into 3 distinct subpopulations based on their locality and function (subsarcolemmal, myofibrillar, and perinuclear). Although the mitochondria are arranged differently, emerging data supports a role for the mitochondrial shaping proteins in cardiac health and disease. Interestingly, in the adult heart, it appears that the pleiotropic effects of the mitochondrial fusion proteins, Mfn2 (endoplasmic reticulum-tethering, mitophagy) and OPA1 (cristae remodeling, regulation of apoptosis, and energy production) may play more important roles than their pro-fusion effects. In this review article, we provide an overview of the mitochondrial fusion and fission proteins in the adult heart, and highlight their roles as novel therapeutic targets for treating cardiac disease.
View details for DOI 10.1007/s10557-016-6710-1
View details for PubMedID 28190190
Regulation of Sema3c and the Interaction between Cardiac Neural Crest and Second Heart Field during Outflow Tract Development.
2017; 7 (1): 6771
The cardiac neural crest cells (cNCCs) and the second heart field (SHF) play key roles in development of the cardiac outflow tract (OFT) for establishment of completely separated pulmonary and systemic circulations in vertebrates. A neurovascular guiding factor, Semaphorin 3c (Sema3c), is required for the development of the OFT, however, its regulation of the interaction between cNCCs and SHF remains to be determined. Here, we show that a Sema3c is a candidate that mediates interaction between cNCCs and the SHF during development of the OFT. Foxc1/c2 directly activates the transcription of Sema3c in the OFT, whereas, a hypomorph of Tbx1, a key SHF transcription factor, resulted in the ectopic expression of Sema3c in the pharyngeal arch region. Fgf8, a downstream secreted factor of Tbx1, inhibited the expression of Sema3c in cNCCs via activation of ERK1/2 signaling. Blocking of FGF8 caused ectopic expression of SEMA3C and a migration defect of cNCCs, resulting in abnormal chick pharyngeal arch development. These results suggest that proper spatio-temporal expression of Sema3c, regulated positively by Foxc1/c2 and negatively by the Tbx1-Fgf8 cascade, respectively, is essential for the interaction between cNCCs and the SHF that correctly navigates cNCCs towards the OFT, composed of SHF-derived cells.
View details for DOI 10.1038/s41598-017-06964-9
View details for PubMedID 28754980
Molecular and functional resemblance of differentiated cells derived from isogenic human iPSCs and SCNT-derived ESCs.
Proceedings of the National Academy of Sciences of the United States of America
Patient-specific pluripotent stem cells (PSCs) can be generated via nuclear reprogramming by transcription factors (i.e., induced pluripotent stem cells, iPSCs) or by somatic cell nuclear transfer (SCNT). However, abnormalities and preclinical application of differentiated cells generated by different reprogramming mechanisms have yet to be evaluated. Here we investigated the molecular and functional features, and drug response of cardiomyocytes (PSC-CMs) and endothelial cells (PSC-ECs) derived from genetically relevant sets of human iPSCs, SCNT-derived embryonic stem cells (nt-ESCs), as well as in vitro fertilization embryo-derived ESCs (IVF-ESCs). We found that differentiated cells derived from isogenic iPSCs and nt-ESCs showed comparable lineage gene expression, cellular heterogeneity, physiological properties, and metabolic functions. Genome-wide transcriptome and DNA methylome analysis indicated that iPSC derivatives (iPSC-CMs and iPSC-ECs) were more similar to isogenic nt-ESC counterparts than those derived from IVF-ESCs. Although iPSCs and nt-ESCs shared the same nuclear DNA and yet carried different sources of mitochondrial DNA, CMs derived from iPSC and nt-ESCs could both recapitulate doxorubicin-induced cardiotoxicity and exhibited insignificant differences on reactive oxygen species generation in response to stress condition. We conclude that molecular and functional characteristics of differentiated cells from human PSCs are primarily attributed to the genetic compositions rather than the reprogramming mechanisms (SCNT vs. iPSCs). Therefore, human iPSCs can replace nt-ESCs as alternatives for generating patient-specific differentiated cells for disease modeling and preclinical drug testing.
View details for DOI 10.1073/pnas.1708991114
View details for PubMedID 29203658
Telomere shortening and metabolic compromise underlie dystrophic cardiomyopathy.
Proceedings of the National Academy of Sciences of the United States of America
Duchenne muscular dystrophy (DMD) is an incurable X-linked genetic disease that is caused by a mutation in the dystrophin gene and affects one in every 3,600 boys. We previously showed that long telomeres protect mice from the lethal cardiac disease seen in humans with the same genetic defect, dystrophin deficiency. By generating the mdx(4cv)/mTR(G2) mouse model with "humanized" telomere lengths, the devastating dilated cardiomyopathy phenotype seen in patients with DMD was recapitulated. Here, we analyze the degenerative sequelae that culminate in heart failure and death in this mouse model. We report progressive telomere shortening in developing mouse cardiomyocytes after postnatal week 1, a time when the cells are no longer dividing. This proliferation-independent telomere shortening is accompanied by an induction of a DNA damage response, evident by p53 activation and increased expression of its target gene p21 in isolated cardiomyocytes. The consequent repression of Pgc1α/β leads to impaired mitochondrial biogenesis, which, in conjunction with the high demands of contraction, leads to increased oxidative stress and decreased mitochondrial membrane potential. As a result, cardiomyocyte respiration and ATP output are severely compromised. Importantly, treatment with a mitochondrial-specific antioxidant before the onset of cardiac dysfunction rescues the metabolic defects. These findings provide evidence for a link between short telomere length and metabolic compromise in the etiology of dilated cardiomyopathy in DMD and identify a window of opportunity for preventive interventions.
View details for PubMedID 27799523
Allogeneic Mesenchymal Stromal Cells Overexpressing Mutant Human Hypoxia-Inducible Factor 1-a (HIF1-a) in an Ovine Model of Acute Myocardial Infarction.
Journal of the American Heart Association
2016; 5 (7)
Bone marrow mesenchymal stromal cells (BMMSCs) are cardioprotective in acute myocardial infarction (AMI) because of release of paracrine angiogenic and prosurvival factors. Hypoxia-inducible factor 1-α (HIF1-α), rapidly degraded during normoxia, is stabilized during ischemia and upregulates various cardioprotective genes. We hypothesized that BMMSCs engineered to overexpress mutant, oxygen-resistant HIF1-α would confer greater cardioprotection than nontransfected BMMSCs in sheep with AMI.Allogeneic BMMSCs transfected with a minicircle vector encoding mutant HIF1-α (BMMSC-HIF) were injected in the peri-infarct of sheep (n=6) undergoing coronary occlusion. Over 2 months, infarct volume measured by cardiac magnetic resonance (CMR) imaging decreased by 71.7±1.3% (P<0.001), and left ventricular (LV) percent ejection fraction (%EF) increased near 2-fold (P<0.001) in the presence of markedly decreased end-systolic volume. Sheep receiving nontransfected BMMSCs (BMMSC; n=6) displayed less infarct size limitation and percent LVEF improvement, whereas in placebo-treated animals (n=6), neither parameters changed over time. HIF1-α-transfected BMMSCs (BMMSC-HIF) induced angio-/arteriogenesis and decreased apoptosis by HIF1-mediated overexpression of erythropoietin, inducible nitrous oxide synthase, vascular endothelial growth factor, and angiopoietin-1. Cell tracking using paramagnetic iron nanoparticles in 12 additional sheep revealed enhanced long-term retention of BMMSC-HIF.Intramyocardial delivery of BMMSC-HIF reduced infarct size and improved LV systolic performance compared to BMMSC, attributed to increased neovascularization and cardioprotective effects induced by HIF1-mediated overexpression of paracrine factors and enhanced retention of injected cells. Given the safety of the minicircle vector and the feasibility of BMMSCs for allogeneic application, this treatment may be potentially useful in the clinic.
View details for DOI 10.1161/JAHA.116.003714
View details for PubMedID 27385426
Human induced pluripotent stem cell-derived cardiomyocytes recapitulate the predilection of breast cancer patients to doxorubicin-induced cardiotoxicity
2016; 22 (5): 547-556
Doxorubicin is an anthracycline chemotherapy agent effective in treating a wide range of malignancies, but it causes a dose-related cardiotoxicity that can lead to heart failure in a subset of patients. At present, it is not possible to predict which patients will be affected by doxorubicin-induced cardiotoxicity (DIC). Here we demonstrate that patient-specific human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) can recapitulate the predilection to DIC of individual patients at the cellular level. hiPSC-CMs derived from individuals with breast cancer who experienced DIC were consistently more sensitive to doxorubicin toxicity than hiPSC-CMs from patients who did not experience DIC, with decreased cell viability, impaired mitochondrial and metabolic function, impaired calcium handling, decreased antioxidant pathway activity, and increased reactive oxygen species production. Taken together, our data indicate that hiPSC-CMs are a suitable platform to identify and characterize the genetic basis and molecular mechanisms of DIC.
View details for DOI 10.1038/nm.4087
View details for Web of Science ID 000375514000018
View details for PubMedID 27089514
Comparison of Magnetic Resonance Imaging and Serum Biomarkers for Detection of Human Pluripotent Stem Cell-Derived Teratomas
STEM CELL REPORTS
2016; 6 (2): 176-187
The use of cells derived from pluripotent stem cells (PSCs) for regenerative therapies confers a considerable risk for neoplastic growth and teratoma formation. Preclinical and clinical assessment of such therapies will require suitable monitoring strategies to understand and mitigate these risks. Here we generated human-induced pluripotent stem cells (iPSCs), selected clones that continued to express reprogramming factors after differentiation into cardiomyocytes, and transplanted these cardiomyocytes into immunocompromised rat hearts post-myocardial infarction. We compared magnetic resonance imaging (MRI), cardiac ultrasound, and serum biomarkers for their ability to delineate teratoma formation and growth. MRI enabled the detection of teratomas with a volume >8 mm(3). A combination of three plasma biomarkers (CEA, AFP, and HCG) was able to detect teratomas with a volume >17 mm(3) and with a sensitivity of more than 87%. Based on our findings, a combination of serum biomarkers with MRI screening may offer the highest sensitivity for teratoma detection and tracking.
View details for DOI 10.1016/j.stemcr.2015.12.008
View details for Web of Science ID 000369726500003
View details for PubMedID 26777057
Parkinson's disease proteins: Novel mitochondrial targets for cardioprotection.
Pharmacology & therapeutics
2015; 156: 34-43
Ischemic heart disease (IHD) is the leading cause of death and disability worldwide. Therefore, novel therapeutic targets for protecting the heart against acute ischemia-reperfusion injury (IRI) are required to attenuate cardiomyocyte death, preserve myocardial function, and prevent the onset of heart failure. In this regard, a specific group of mitochondrial proteins, which have been linked to familial forms of Parkinson's disease (PD), may provide novel therapeutic targets for cardioprotection. In dopaminergic neurons of the substantial nigra, these PD proteins, which include Parkin, PINK1, DJ-1, LRRK2, and α-synuclein, play essential roles in preventing cell death - through maintaining normal mitochondrial function, protecting against oxidative stress, mediating mitophagy, and preventing apoptosis. These rare familial forms of PD may therefore provide important insights into the pathophysiology underlying mitochondrial dysfunction and the development of PD. Interestingly, these PD proteins are also present in the heart, but their role in myocardial health and disease is not clear. In this article we review the role of these PD proteins in the heart and explore their potential as novel mitochondrial targets for cardioprotection.
View details for DOI 10.1016/j.pharmthera.2015.10.005
View details for PubMedID 26481155
- Assessment of the Radiation Effects of Cardiac CT Angiography Using Protein and Genetic Biomarkers JACC-CARDIOVASCULAR IMAGING 2015; 8 (8): 873-884
MicroRNA-mediated regulation of differentiation and trans-differentiation in stem cells
ADVANCED DRUG DELIVERY REVIEWS
2015; 88: 3-15
MicroRNAs (miRNAs) are key components of a broadly conserved post-transcriptional mechanism that controls gene expression by targeting mRNAs. miRNAs regulate diverse biological processes, including the growth and differentiation of stem cells as well as the regulation of both endogenous tissue repair that has critical implications in the development of regenerative medicine approaches. In this review, we first describe key features of miRNA biogenesis and their role in regulating self-renewal, and then discuss the involvement of miRNAs in the determination of cell fate decisions. We highlight the role of miRNAs in the emergent field of reprogramming and trans-differentiation of somatic cells that could further our understanding of miRNA biology and regenerative medicine applications. Finally, we describe potential techniques for proper delivery of miRNAs in target cells.
View details for DOI 10.1016/j.addr.2015.04.004
View details for Web of Science ID 000358628200002
- Exosomes as potential alternatives to stem cell therapy in mediating cardiac regeneration. Circulation research 2015; 117 (1): 7-9
Pravastatin reverses obesity-induced dysfunction of induced pluripotent stem cell-derived endothelial cells via a nitric oxide-dependent mechanism.
European heart journal
2015; 36 (13): 806-816
High-fat diet-induced obesity (DIO) is a major contributor to type II diabetes and micro- and macro-vascular complications leading to peripheral vascular disease (PVD). Metabolic abnormalities of induced pluripotent stem cell-derived endothelial cells (iPSC-ECs) from obese individuals could potentially limit their therapeutic efficacy for PVD. The aim of this study was to compare the function of iPSC-ECs from normal and DIO mice using comprehensive in vitro and in vivo assays.Six-week-old C57Bl/6 mice were fed with a normal or high-fat diet. At 24 weeks, iPSCs were generated from tail tip fibroblasts and differentiated into iPSC-ECs using a directed monolayer approach. In vitro functional analysis revealed that iPSC-ECs from DIO mice had significantly decreased capacity to form capillary-like networks, diminished migration, and lower proliferation. Microarray and ELISA confirmed elevated apoptotic, inflammatory, and oxidative stress pathways in DIO iPSC-ECs. Following hindlimb ischaemia, mice receiving intramuscular injections of DIO iPSC-ECs had significantly decreased reperfusion compared with mice injected with control healthy iPSC-ECs. Hindlimb sections revealed increased muscle atrophy and presence of inflammatory cells in mice receiving DIO iPSC-ECs. When pravastatin was co-administered to mice receiving DIO iPSC-ECs, a significant increase in reperfusion was observed; however, this beneficial effect was blunted by co-administration of the nitric oxide synthase inhibitor, N(ω)-nitro-l-arginine methyl ester.This is the first study to provide evidence that iPSC-ECs from DIO mice exhibit signs of endothelial dysfunction and have suboptimal efficacy following transplantation in a hindlimb ischaemia model. These findings may have important implications for future treatment of PVD using iPSC-ECs in the obese population.
View details for DOI 10.1093/eurheartj/ehu411
View details for PubMedID 25368203
- Response to letter regarding article, "Cross talk of combined gene and cell therapy in ischemic heart disease: role of exosomal microRNA transfer". Circulation 2015; 131 (12)
Variable activation of the DNA damage response pathways in patients undergoing single-photon emission computed tomography myocardial perfusion imaging.
Circulation. Cardiovascular imaging
2015; 8 (2)
Although single-photon emission computed tomography myocardial perfusion imaging (SPECT MPI) has improved the diagnosis and risk stratification of patients with suspected coronary artery disease, it remains a primary source of low-dose radiation exposure for cardiac patients. To determine the biological effects of low-dose radiation from SPECT MPI, we measured the activation of the DNA damage response pathways using quantitative flow cytometry and single-cell gene expression profiling.Blood samples were collected from patients before and after SPECT MPI (n=63). Overall, analysis of all recruited patients showed no marked differences in the phosphorylation of proteins (H2AX, protein 53, and ataxia telangiectasia mutated) after SPECT. The majority of patients also had either downregulated or unchanged expression in DNA damage response genes at both 24 and 48 hours post-SPECT. Interestingly, a small subset of patients with increased phosphorylation had significant upregulation of genes associated with DNA damage, whereas those with no changes in phosphorylation had significant downregulation or no difference, suggesting that some patients may potentially be more sensitive to low-dose radiation exposure.Our findings showed that SPECT MPI resulted in a variable activation of the DNA damage response pathways. Although only a small subset of patients had increased protein phosphorylation and elevated gene expression postimaging, continued care should be taken to reduce radiation exposure to both the patients and operators.
View details for DOI 10.1161/CIRCIMAGING.114.002851
View details for PubMedID 25609688
View details for PubMedCentralID PMC4354894
Effect of human donor cell source on differentiation and function of cardiac induced pluripotent stem cells.
Journal of the American College of Cardiology
2014; 64 (5): 436-448
Human-induced pluripotent stem cells (iPSCs) are a potentially unlimited source for generation of cardiomyocytes (iPSC-CMs). However, current protocols for iPSC-CM derivation face several challenges, including variability in somatic cell sources and inconsistencies in cardiac differentiation efficiency.This study aimed to assess the effect of epigenetic memory on differentiation and function of iPSC-CMs generated from somatic cell sources of cardiac versus noncardiac origins.Cardiac progenitor cells (CPCs) and skin fibroblasts from the same donors were reprogrammed into iPSCs and differentiated into iPSC-CMs via embryoid body and monolayer-based differentiation protocols.Differentiation efficiency was found to be higher in CPC-derived iPSC-CMs (CPC-iPSC-CMs) than in fibroblast-derived iPSC-CMs (Fib-iPSC-CMs). Gene expression analysis during cardiac differentiation demonstrated up-regulation of cardiac transcription factors in CPC-iPSC-CMs, including NKX2-5, MESP1, ISL1, HAND2, MYOCD, MEF2C, and GATA4. Epigenetic assessment revealed higher methylation in the promoter region of NKX2-5 in Fib-iPSC-CMs compared with CPC-iPSC-CMs. Epigenetic differences were found to dissipate with increased cell passaging, and a battery of in vitro assays revealed no significant differences in their morphological and electrophysiological properties at early passage. Finally, cell delivery into a small animal myocardial infarction model indicated that CPC-iPSC-CMs and Fib-iPSC-CMs possess comparable therapeutic capabilities in improving functional recovery in vivo.This is the first study to compare differentiation of iPSC-CMs from human CPCs versus human fibroblasts from the same donors. The authors demonstrate that although epigenetic memory improves differentiation efficiency of cardiac versus noncardiac somatic cell sources in vitro, it does not contribute to improved functional outcome in vivo.
View details for DOI 10.1016/j.jacc.2014.04.056
View details for PubMedID 25082575
View details for PubMedCentralID PMC4134946
Development of Poly(ß-amino ester)-Based Biodegradable Nanoparticles for Nonviral Delivery of Minicircle DNA.
2013; 7 (8): 7241-7250
Gene therapy provides a powerful tool for regulating cellular processes and tissue repair. Minicircle (MC) DNA are supercoiled DNA molecules free of bacterial plasmid backbone elements and have been reported to enhance prolonged gene expression compared to conventional plasmids. Despite the great promise of MC DNA for gene therapy, methods for safe and efficient MC DNA delivery remain lacking. To overcome this bottleneck, here we report the development of a poly(β-amino ester) (PBAE)-based, biodegradable nanoparticulate platform for efficient delivery of MC DNA driven by a Ubc promoter in vitro and in vivo. By synthesizing and screening a small library of 18 PBAE polymers with different backbone and end-group chemistry, we identified lead cationic PBAE structures that can complex with minicircle DNA to form nanoparticles, and delivery efficiency can be further modulated by tuning PBAE chemistry. Using human embryonic kidney 293 cells and mouse embryonic fibroblasts as model cell types, we identified a few PBAE polymers that allow efficient MC delivery at levels that are comparable or even surpassing Lipofectamine 2000. The biodegradable nature of PBAE-based nanoparticles facilitates in vivo applications and clinical translation. When injected via intraperitoneal route in vivo, MC alone resulted in high transgene expression, and a lead PBAE/MC nanoparticle formulation achieved a further 2-fold increase in protein expression compared to MC alone. Together, our results highlight the promise of PBAE-based nanoparticles as promising nonviral gene carriers for MC delivery, which may provide a valuable tool for broad applications of MC DNA-based gene therapy.
View details for DOI 10.1021/nn402657d
View details for PubMedID 23837668
- Cortical bone-derived stem cells: a novel class of cells for myocardial protection. Circulation research 2013; 113 (5): 480-483
Hypoxia-inducible factor as a therapeutic target for cardioprotection
PHARMACOLOGY & THERAPEUTICS
2012; 136 (1): 69-81
Hypoxia inducible factor (HIF) is an oxygen-sensitive transcription factor that enables aerobic organisms to adapt to hypoxia. This is achieved through the transcriptional activation of up to 200 genes, many of which are critical to cell survival. Under conditions of normoxia, the hydroxylation of HIF by prolyl hydroxylase domain-containing (PHD) enzymes targets it for polyubiquitination and proteosomal degradation by the von Hippel-Lindau protein (VHL). However, under hypoxic conditions, PHD activity is inhibited, thereby allowing HIF to accumulate and translocate to the nucleus, where it binds to the hypoxia-responsive element sequences of target gene promoters. Experimental studies suggest that HIF may act as a mediator of ischemic preconditioning, and that the genetic or pharmacological stabilization of HIF under normoxic conditions, may protect the heart against the detrimental effects of acute ischemia-reperfusion injury. The mechanisms underlying the cardioprotective effect of HIF are unclear, but it may be attributed to the transcriptional activation of genes associated with cardioprotection such as erythropoietin, heme oxygenase-1, and inducible nitric oxide synthase or it may be due to reprogramming of cell metabolism. In this review article, we highlight the role of HIF in mediating both adaptive and pathological processes in the heart, as well as focusing on the therapeutic potential of the HIF-signaling pathway as a target for cardioprotection.
View details for DOI 10.1016/j.pharmthera.2012.07.005
View details for Web of Science ID 000309034600006
View details for PubMedID 22800800
Leptin-induced cardioprotection involves JAK/STAT signaling that may be linked to the mitochondrial permeability transition pore
AMERICAN JOURNAL OF PHYSIOLOGY-HEART AND CIRCULATORY PHYSIOLOGY
2010; 299 (4): H1265-H1270
Leptin-induced protection against myocardial ischemia-reperfusion (I/R) injury involves the activation of the reperfusion injury salvage kinase pathway, incorporating phosphatidylinositol 3-kinase-Akt/protein kinase B and p44/42 MAPK, and the inhibition of the mitochondrial permeability transition pore (MPTP). Recently published data indicate that the JAK/STAT signaling pathway, which mediates the metabolic actions of leptin, also plays a pivotal role in cardioprotection. Consequently, in the present study we considered the possibility that JAK/STAT signaling linked to the MPTP may be involved in modulating the cardioprotective actions of leptin. Employing rat in vitro models (Langendorff-perfused hearts and cardiomyocytes) of I/R injury, we investigated the actions of leptin (10 nM), administered at reperfusion, in the presence or absence of the JAK2 inhibitor, AG-490 (5 μM). Leptin reduced infarct size significantly (control, 60.05 ± 7.41% vs. leptin treated, 29.9 ± 3.24%, P < 0.05), protection being abolished by AG-490. Time course studies revealed that leptin caused a 171% (P < 0.001) increase in STAT3/tyrosine-705 phosphorylation at 2.5 min reperfusion; however, increases were not seen at 5, 10, 15, or 30 min reperfusion. Contrasting with STAT3, Akt/serine-473 phosphorylation was not significantly increased until 15 min into the reperfusion phase (140%, P < 0.05). AG-490 blocked the leptin-induced rise in STAT3 phosphorylation seen at 2.5 min reperfusion but did not influence Akt/serine-473 phosphorylation at 15 min. Leptin reduced the MPTP opening (P < 0.001), which was blocked by AG-490. This is the first study to yield evidence that JAK/STAT signaling linked to the MPTP plays a role in leptin-induced cardioprotection. Under the experimental conditions employed, STAT3 phosphorylation appears to have occurred earlier during reperfusion than that of Akt. Further research into the interactions between these two signaling pathways in the setting of I/R injury is, however, required.
View details for DOI 10.1152/ajpheart.00092.2010
View details for Web of Science ID 000283857300034
View details for PubMedID 20656889
Mitochondrial cyclophilin-D as a critical mediator of ischaemic preconditioning
2010; 88 (1): 67-74
It has been suggested that mitochondrial reactive oxygen species (ROS), Akt and Erk1/2 and more recently the mitochondrial permeability transition pore (mPTP) may act as mediators of ischaemic preconditioning (IPC), although the actual interplay between these mediators is unclear. The aim of the present study is to determine whether the cyclophilin-D (CYPD) component of the mPTP is required by IPC to generate mitochondrial ROS and subsequently activate Akt and Erk1/2.Mice lacking CYPD (CYPD-/-) and B6Sv129 wild-type (WT) mice were used throughout. We have demonstrated that under basal conditions, non-pathological mPTP opening occurs (indicated by the percent reduction in mitochondrial calcein fluorescence). This effect was greater in WT cardiomyocytes compared with CYPD-/- ones (53 ± 2% WT vs. 17 ± 3% CYPD-/-; P < 0.01) and was augmented by hypoxic preconditioning (HPC) (70 ± 9% WT vs. 56 ± 1% CYPD-/-; P < 0.01). HPC reduced cell death following simulated ischaemia-reperfusion injury in WT (23.2 ± 3.5% HPC vs. 43.7 ± 3.2% WT; P < 0.05) but not CYPD-/- cardiomyocytes (19.6 ± 1.4% HPC vs. 24.4 ± 2.6% control; P > 0.05). HPC generated mitochondrial ROS in WT (four-fold increase; P < 0.05) but not CYPD-/- cardiomyocytes. HPC induced significant Akt phosphorylation in WT cardiomyocytes (two-fold increase; P < 0.05), an effect which was abrogated by ciclosporin-A (a CYPD inhibitor) and N-2-mercaptopropionyl glycine (a ROS scavenger). Finally, in vivo IPC of adult murine hearts resulted in significant phosphorylation of Akt and Erk1/2 in WT but not CYPD-/- hearts.The CYPD component of the mPTP is required by IPC to generate mitochondrial ROS and phosphorylate Akt and Erk1/2, major steps in the IPC signalling pathway.
View details for DOI 10.1093/cvr/cvq113
View details for Web of Science ID 000281714600010
View details for PubMedID 20400621