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1.  Epigenetic regulation of cardiac myocyte differentiation† 
Frontiers in Genetics  2014;5:375.
Cardiac myocytes (CMs) proliferate robustly during fetal life but withdraw permanently from the cell cycle soon after birth and undergo terminal differentiation. This cell cycle exit is associated with the upregulation of a host of adult cardiac-specific genes. The vast majority of adult CMs (ACMs) do not reenter cell cycle even if subjected to mitogenic stimuli. The basis for this irreversible cell cycle exit is related to the stable silencing of cell cycle genes specifically involved in the progression of G2/M transition and cytokinesis. Studies have begun to clarify the molecular basis for this stable gene repression and have identified epigenetic and chromatin structural changes in this process. In this review, we summarize the current understanding of epigenetic regulation of CM cell cycle and cardiac-specific gene expression with a focus on histone modifications and the role of retinoblastoma family members.
PMCID: PMC4219506  PMID: 25408700
cardiac myocyte; proliferation; differentiation; heterochromatin; histone modification; retinoblastoma protein
2.  Dietary restriction attenuates the accelerated aging phenotype of Sod1−/− mice 
Dietary restriction is a powerful aging intervention that extends the life span of diverse biological species ranging from yeast to invertebrates to mammals, and it has been argued that the anti-aging action of dietary restriction occurs through reduced oxidative stress/damage. Using Sod1−/− mice, which have previously been shown to have increased levels of oxidative stress associated with a shorter life span and a high incidence of neoplasia, we were able to test directly the ability of dietary restriction to reverse an aging phenotype due to increased oxidative stress/damage. We found that dietary restriction increased the life span of Sod1−/− mice 30%, returning it to that of wild type, control mice fed ad libitum. Oxidative damage in Sod1−/− mice was markedly reduced by dietary restriction, as indicated by a reduction in liver and brain F2-isoprostanes, a marker of lipid peroxidation. Analysis of end of life pathology showed that dietary restriction significantly reduced the overall incidence of pathological lesions in the Sod1−/− mice fed the dietary restricted-diet compared to Sod1−/− mice fed ad libitum, including the incidence of lymphoma (27 vs 5%) and overall liver pathology. In addition to reduced incidence of overall and liver specific pathology, the burden and severity of both neoplastic and non-neoplastic lesions was also significantly reduced in the Sod1−/− mice fed the dietary restricted-diet. These data demonstrate that dietary restriction can significantly attenuate the accelerated aging phenotype observed in Sod1−/− mice that arises from increased oxidative stress/damage.
PMCID: PMC3696984  PMID: 23459073
dietary restriction; aging oxidative stress; CuZnSOD
3.  The Lack of CuZnSOD Leads to Impaired Neurotransmitter Release, Neuromuscular Junction Destabilization and Reduced Muscle Strength in Mice 
PLoS ONE  2014;9(6):e100834.
Elevated reactive oxygen species (ROS) production and ROS-dependent protein damage is a common observation in the pathogenesis of many muscle wasting disorders, including sarcopenia. However, the contribution of elevated ROS levels to –a breakdown in neuromuscular communication and muscle atrophy remains unknown. In this study, we examined a copper zinc superoxide dismutase [CuZnSOD (Sod1)] knockout mouse (Sod1−/−), a mouse model of elevated oxidative stress that exhibits accelerated loss of muscle mass, which recapitulates many phenotypes of sarcopenia as early as 5 months of age. We found that young adult Sod1−/− mice display a considerable reduction in hind limb skeletal muscle mass and strength when compared to age-matched wild-type mice. These changes are accompanied by gross alterations in neuromuscular junction (NMJ) morphology, including reduced occupancy of the motor endplates by axons, terminal sprouting and axon thinning and irregular swelling. Surprisingly however, the average density of acetylcholine receptors in endplates is preserved. Using in vivo electromyography and ex vivo electrophysiological studies of hind limb muscles in Sod1−/− mice, we found that motor axons innervating the extensor digitorum longus (EDL) and gastrocnemius muscles release fewer synaptic vesicles upon nerve stimulation. Recordings from individually identified EDL NMJs show that reductions in neurotransmitter release are apparent in the Sod1−/− mice even when endplates are close to fully innervated. However, electrophysiological properties, such as input resistance, resting membrane potential and spontaneous neurotransmitter release kinetics (but not frequency) are similar between EDL muscles of Sod1−/− and wild-type mice. Administration of the potassium channel blocker 3,4-diaminopyridine, which broadens the presynaptic action potential, improves both neurotransmitter release and muscle strength. Together, these results suggest that ROS-associated motor nerve terminal dysfunction is a contributor to the observed muscle changes in Sod1−/− mice.
PMCID: PMC4074103  PMID: 24971750
4.  Targeted MicroRNA Interference Promotes Postnatal Cardiac Cell Cycle Re-Entry 
Mammalian heart cells undergo a marked reduction in proliferative activity shortly after birth, and thereafter grow predominantly by hypertrophy. Our understanding of the molecular mechanisms underlying cardiac maturation and senescence is based largely on studies at the whole-heart level. Here, we investigate the molecular basis of the acquired quiescence of purified neonatal and adult cardiomyocytes, and use microRNA interference as a novel strategy to promote cardiomyocyte cell cycle re-entry. Expression of cyclins and cyclin-dependent kinases (CDKs) and positive modulators were down-regulated, while CDK inhibitors and negative cell cycle modulators were up-regulated during postnatal maturation of cardiomyocytes. The expression pattern of microRNAs also changed dramatically, including increases in miR-29a, miR-30a and miR-141. Treatment of neonatal cardiomyocytes with miRNA inhibitors anti-miR-29a, anti-miR-30a, and antimiR-141 resulted in more cycling cells and enhanced expression of Cyclin A2 (CCNA2). Thus, targeted microRNA interference can reactivate postnatal cardiomyocyte proliferation.
PMCID: PMC4048036  PMID: 24910852
Cell cycle; Cardiomyocytes; Cyclins; microRNA; Western blot
5.  THY-1 Receptor Expression Differentiates Cardiosphere-Derived Cells with Divergent Cardiogenic Differentiation Potential 
Stem Cell Reports  2014;2(5):576-591.
Despite over a decade of intense research, the identity and differentiation potential of human adult cardiac progenitor cells (aCPC) remains controversial. Cardiospheres have been proposed as a means to expand aCPCs in vitro, but the identity of the progenitor cell within these 3D structures is unknown. We show that clones derived from cardiospheres could be subdivided based on expression of thymocyte differentiation antigen 1 (THY-1/CD90) into two distinct populations that exhibit divergent cardiac differentiation potential. One population, which is CD90+, expressed markers consistent with a mesenchymal/myofibroblast cell. The second clone type was CD90− and could form mature, functional myocytes with sarcomeres albeit at a very low rate. These two populations of cardiogenic clones displayed distinct cell surface markers and unique transcriptomes. Our study suggests that a rare aCPC exists in cardiospheres along with a mesenchymal/myofibroblast cell, which demonstrates incomplete cardiac myocyte differentiation.
•The majority of human adult CS-derived cardiomyocytes do not form sarcomeres•There is little overlap between the C-KIT and NKX2.5 cell populations in human CS•CD90 distinguishes two populations that exhibit divergent cardiogenic potential•CD90neg CS cells can differentiate into mature cardiomyocytes at a very low rate
Cardiospheres (CSs) have been reported to contain an adult cardiac progenitor cell (aCPC) with multilineage differentiation potential. MacLellan and colleagues show that CS-derived cells differentiate robustly into endothelial and smooth muscle cells but predominantly form immature cardiomyocytes. CD90 expression distinguishes CS-derived cells with a mesenchymal cell phenotype with incomplete cardiomyocyte differentiation (CD90+) and a CD90− population that could give rise to mature cardiomyocytes, although infrequently.
PMCID: PMC4050474  PMID: 24936447
6.  Combined treatment of rapamycin and dietary restriction has a larger effect on the transcriptome and metabolome of liver 
Aging Cell  2013;13(2):311-319.
Rapamycin (Rapa) and dietary restriction (DR) have consistently been shown to increase lifespan. To investigate whether Rapa and DR affect similar pathways in mice, we compared the effects of feeding mice ad libitum (AL), Rapa, DR, or a combination of Rapa and DR (Rapa + DR) on the transcriptome and metabolome of the liver. The principal component analysis shows that Rapa and DR are distinct groups. Over 2500 genes are significantly changed with either Rapa or DR when compared with mice fed AL; more than 80% are unique to DR or Rapa. A similar observation was made when genes were grouped into pathways; two-thirds of the pathways were uniquely changed by DR or Rapa. The metabolome shows an even greater difference between Rapa and DR; no metabolites in Rapa-treated mice were changed significantly from AL mice, whereas 173 metabolites were changed in the DR mice. Interestingly, the number of genes significantly changed by Rapa + DR when compared with AL is twice as large as the number of genes significantly altered by either DR or Rapa alone. In summary, the global effects of DR or Rapa on the liver are quite different and a combination of Rapa and DR results in alterations in a large number of genes and metabolites that are not significantly changed by either manipulation alone, suggesting that a combination of DR and Rapa would be more effective in extending longevity than either treatment alone.
PMCID: PMC3989927  PMID: 24304444
dietary restriction; metabolome; rapamycin; transcriptome
7.  Molecular characterization of heterogeneous mesenchymal stem cells with single-cell transcriptomes 
Biotechnology advances  2012;31(2):312-317.
Mesenchymal stem cells (MSC) are heterogeneous cell populations with promising therapeutic potentials in regenerative medicine. The therapeutic values of MSC in various clinical situations have been reported. Clonal assays (expansion of MSC from a single cell) demonstrated that multiple types of cells with different developmental potential exist in a MSC population. Due to the heterogeneous nature of MSC, molecular characterization of MSC in the absence of known biomarkers is a challenge for cell therapy with MSC. Here, we review potential therapeutic applications of MSC and discuss a systematic approach for molecular characterization of heterogeneous cell population using single-cell transcriptome analysis. Differentiation/maturation of cells is orchestrated by sequential expression of a series of genes within a cell. Therefore, single-cell mRNA expression (transcriptome) profiles from consecutive developmental stages are more similar than those from disparate stages. Bioinformatic analysis can cluster single-cell transcriptome profiles from consecutive developmental stages into a dendrogram based on the similarity matrix of these profiles.
Because a single-cell is an ultimately “pure” sample in expression profiling, these dendrograms can be used to classify individual cells into molecular subpopulations within a heterogeneous cell population without known biomarkers. This approach is especially powerful in studying cell populations with little molecular information and few known biomarkers, for example the MSC populations. The molecular understanding will provide novel targets for manipulating MSC differentiation with small molecules and other drugs to enable safer and more effective therapeutic applications of MSC.
PMCID: PMC3767306  PMID: 23266308
Single-cell transcriptome; Heterogeneity; Mesenchymal stem cells
8.  Mice Fed Rapamycin Have an Increase in Lifespan Associated with Major Changes in the Liver Transcriptome 
PLoS ONE  2014;9(1):e83988.
Rapamycin was found to increase (11% to 16%) the lifespan of male and female C57BL/6J mice most likely by reducing the increase in the hazard for mortality (i.e., the rate of aging) term in the Gompertz mortality analysis. To identify the pathways that could be responsible for rapamycin's longevity effect, we analyzed the transcriptome of liver from 25-month-old male and female mice fed rapamycin starting at 4 months of age. Few changes (<300 transcripts) were observed in transcriptome of rapamycin-fed males; however, a large number of transcripts (>4,500) changed significantly in females. Using multidimensional scaling and heatmap analyses, the male mice fed rapamycin were found to segregate into two groups: one group that is almost identical to control males (Rapa-1) and a second group (Rapa-2) that shows a change in gene expression (>4,000 transcripts) with more than 60% of the genes shared with female mice fed Rapa. Using ingenuity pathway analysis, 13 pathways were significantly altered in both Rapa-2 males and rapamycin-fed females with mitochondrial function as the most significantly changed pathway. Our findings show that rapamycin has a major effect on the transcriptome and point to several pathways that would likely impact the longevity.
PMCID: PMC3883653  PMID: 24409289
9.  Novel Biomarkers of Arterial and Venous Ischemia in Microvascular Flaps 
PLoS ONE  2013;8(8):e71628.
The field of reconstructive microsurgery is experiencing tremendous growth, as evidenced by recent advances in face and hand transplantation, lower limb salvage after trauma, and breast reconstruction. Common to all of these procedures is the creation of a nutrient vascular supply by microsurgical anastomosis between a single artery and vein. Complications related to occluded arterial inflow and obstructed venous outflow are not uncommon, and can result in irreversible tissue injury, necrosis, and flap loss. At times, these complications are challenging to clinically determine. Since early intervention with return to the operating room to re-establish arterial inflow or venous outflow is key to flap salvage, the accurate diagnosis of early stage complications is essential. To date, there are no biochemical markers or serum assays that can predict these complications. In this study, we utilized a rat model of flap ischemia in order to identify the transcriptional signatures of venous congestion and arterial ischemia. We found that the critical ischemia time for the superficial inferior epigastric fasciocutaneus flap was four hours and therefore performed detailed analyses at this time point. Histolgical analysis confirmed significant differences between arterial and venous ischemia. The transcriptome of ischemic, congested, and control flap tissues was deciphered by performing Affymetrix microarray analysis and verified by qRT-PCR. Principal component analysis revealed that arterial ischemia and venous congestion were characterized by distinct transcriptomes. Arterial ischemia and venous congestion was characterized by 408 and 1536>2-fold differentially expressed genes, respectively. qRT-PCR was used to identify five candidate genes Prol1, Muc1, Fcnb, Il1b, and Vcsa1 to serve as biomarkers for flap failure in both arterial ischemia and venous congestion. Our data suggests that Prol1 and Vcsa1 may be specific indicators of venous congestion and allow clinicians to both diagnose and successfully treat microvascular complications before irreversible tissue damage and flap loss occurs.
PMCID: PMC3743756  PMID: 23977093
10.  Elevated Protein Carbonylation, and Misfolding in Sciatic Nerve from db/db and Sod1−/− Mice: Plausible Link between Oxidative Stress and Demyelination 
PLoS ONE  2013;8(6):e65725.
Diabetic peripheral polyneuropathy is associated with decrements in motor/sensory neuron myelination, nerve conduction and muscle function; however, the mechanisms of reduced myelination in diabetes are poorly understood. Chronic elevation of oxidative stress may be one of the potential determinants for demyelination as lipids and proteins are important structural constituents of myelin and highly susceptible to oxidation. The goal of the current study was to determine whether there is a link between protein oxidation/misfolding and demyelination. We chose two distinct models to test our hypothesis: 1) the leptin receptor deficient mouse (dbdb) model of diabetic polyneuropathy and 2) superoxide dismutase 1 knockout (Sod1−/−) mouse model of in vivo oxidative stress. Both experimental models displayed a significant decrement in nerve conduction, increase in tail distal motor latency as well as reduced myelin thickness and fiber/axon diameter. Further biochemical studies demonstrated that oxidative stress is likely to be a potential key player in the demyelination process as both models exhibited significant elevation in protein carbonylation and alterations in protein conformation. Since peripheral myelin protein 22 (PMP22) is a key component of myelin sheath and has been found mutated and aggregated in several peripheral neuropathies, we predicted that an increase in carbonylation and aggregation of PMP22 may be associated with demyelination in dbdb mice. Indeed, PMP22 was found to be carbonylated and aggregated in sciatic nerves of dbdb mice. Sequence-driven hydropathy plot analysis and in vitro oxidation-induced aggregation of purified PMP22 protein supported the premise for oxidation-dependent aggregation of PMP22 in dbdb mice. Collectively, these data strongly suggest for the first time that oxidation-mediated protein misfolding and aggregation of key myelin proteins may be linked to demyelination and reduced nerve conduction in peripheral neuropathies.
PMCID: PMC3672154  PMID: 23750273
11.  Functional Impairment of Human Resident Cardiac Stem Cells by the Cardiotoxic Antineoplastic Agent Trastuzumab 
Human cardiosphere-derived cells (hCDCs) were exposed to trastuzumab (TZM) to evaluate whether TZM cardiotoxicity involves inhibition of human adult cardiac-derived stem cells, in addition to previously reported direct adverse effects on cardiomyocytes. The results indicated that TZM inhibits the cardiomyogenic and angiogenic capacities of hCDCs in vitro and abrogates the morphological and functional benefits of hCDC transplantation in vivo. Thus, TZM impairs the function of human resident cardiac stem cells, potentially contributing to TZM cardiotoxicity.
Trastuzumab (TZM), a monoclonal antibody against the ERBB2 protein, increases survival in ERBB2-positive breast cancer patients. Its clinical use, however, is limited by cardiotoxicity. We sought to evaluate whether TZM cardiotoxicity involves inhibition of human adult cardiac-derived stem cells, in addition to previously reported direct adverse effects on cardiomyocytes. To test this idea, we exposed human cardiosphere-derived cells (hCDCs), a natural mixture of cardiac stem cells and supporting cells that has been shown to exert potent regenerative effects, to TZM and tested the effects in vitro and in vivo. We found that ERBB2 mRNA and protein are expressed in hCDCs at levels comparable to those in human myocardium. Although clinically relevant concentrations of TZM had no effect on proliferation, apoptosis, or size of the c-kit-positive hCDC subpopulation, in vitro assays demonstrated diminished potential for cardiogenic differentiation and impaired ability to form microvascular networks in TZM-treated cells. The functional benefit of hCDCs injected into the border zone of acutely infarcted mouse hearts was abrogated by TZM: infarcted animals treated with TZM + hCDCs had a lower ejection fraction, thinner infarct scar, and reduced capillary density in the infarct border zone compared with animals that received hCDCs alone (n = 12 per group). Collectively, these results indicate that TZM inhibits the cardiomyogenic and angiogenic capacities of hCDCs in vitro and abrogates the morphological and functional benefits of hCDC transplantation in vivo. Thus, TZM impairs the function of human resident cardiac stem cells, potentially contributing to TZM cardiotoxicity.
PMCID: PMC3659699  PMID: 23197808
Adult stem cells; Cardiac; Chemotherapy; Angiogenesis
12.  Direct comparison of different stem cell types and subpopulations reveals superior paracrine potency and myocardial repair efficacy with cardiosphere-derived cells 
To conduct a direct head-to-head comparison of different stem cell types in vitro for various assays of potency, and in vivo for functional myocardial repair in the same mouse model of myocardial infarction.
Adult stem cells of diverse origins (e.g., bone marrow, fat, heart) and antigenic identity have been studied for repair of the damaged heart, but the relative utility of the various cell types remains unclear.
Human cardiosphere-derived stem cells (CDCs), bone marrow-derived mesenchymal stem cells (BM-MSCs), adipose tissue-derived mesenchymal stem cells (AD-MSCs), and bone marrow mononuclear cells (BM-MNCs) were compared.
CDCs revealed a distinctive phenotype with uniform expression of CD105, partial expression of c-kit and CD90, and negligible expression of hematopoietic markers. In vitro, CDCs showed the greatest myogenic differentiation potency, highest angiogenic potential, and relatively high production of various angiogenic and anti-apoptotic secreted factors. In vivo, injection of CDCs into the infarcted mouse hearts resulted in superior improvement of cardiac function, the highest cell engraftment and myogenic differentiation rates, and the least-abnormal heart morphology 3 weeks after treatment. CDC-treated hearts also exhibited the lowest number of apoptotic cells. The c-kit+ subpopulation purified from CDCs produced lower levels of paracrine factors and inferior functional benefit when compared to unsorted CDCs. To validate the comparison of cells from various human donors, selected results were confirmed in cells of different types derived from individual rats.
CDCs exhibit a balanced profile of paracrine factor production, and, among various comparator cell types/subpopulations, provide the greatest functional benefit in experimental myocardial infarction.
PMCID: PMC3292778  PMID: 22381431
cardiac stem cells; mesenchymal stem cells; myocardial regeneration; paracrine effects
13.  Cardiomyocyte proliferation and progenitor cell recruitment underlie therapeutic regeneration after myocardial infarction in the adult mouse heart 
EMBO Molecular Medicine  2013;5(2):191-209.
Cardiosphere-derived cells (CDCs) have been shown to regenerate infarcted myocardium in patients after myocardial infarction (MI). However, whether the cells of the newly formed myocardium originate from the proliferation of adult cardiomyocytes or from the differentiation of endogenous stem cells remains unknown. Using genetic fate mapping to mark resident myocytes in combination with long-term BrdU pulsing, we investigated the origins of postnatal cardiomyogenesis in the normal, infarcted and cell-treated adult mammalian heart. In the normal mouse heart, cardiomyocyte turnover occurs predominantly through proliferation of resident cardiomyocytes at a rate of ∼1.3–4%/year. After MI, new cardiomyocytes arise from both progenitors as well as pre-existing cardiomyocytes. Transplantation of CDCs upregulates host cardiomyocyte cycling and recruitment of endogenous progenitors, while boosting heart function and increasing viable myocardium. The observed phenomena cannot be explained by cardiomyocyte polyploidization, bi/multinucleation, cell fusion or DNA repair. Thus, CDCs induce myocardial regeneration by differentially upregulating two mechanisms of endogenous cell proliferation.
PMCID: PMC3569637  PMID: 23255322
cardiac regeneration; cardiomyogenesis; cell therapy; fate mapping; myocardial infarction
14.  Safety and Efficacy of Allogeneic Cell Therapy in Infarcted Rats Transplanted with Mismatched Cardiosphere-Derived Cells 
Circulation  2011;125(1):100-112.
Cardiosphere-derived cells (CDCs) are an attractive cell type for tissue regeneration, and autologous CDCs are being tested clinically. However, autologous therapy necessitates patient-specific tissue harvesting and cell processing, with delays to therapy and possible variations in cell potency. The use of allogeneic CDCs, if safe and effective, would obviate such limitations. We compared syngeneic and allogeneic CDC transplantation in rats from immunologically-mismatched inbred strains.
Methods and Results
In vitro, CDCs expressed MHC class I but not class II antigens or B7 costimulatory molecules. In mixed lymphocyte co-cultures, allogeneic CDCs elicited negligible lymphocyte proliferation and inflammatory cytokine secretion. In vivo, syngeneic and allogeneic CDCs survived at similar levels in the infarcted rat heart 1 week after delivery, but few syngeneic (and even fewer allogeneic) CDCs remained at 3 weeks. Allogeneic CDCs induced a transient, mild, local immune reaction in the heart, without histologically-evident rejection or systemic immunogenicity. Improvements in cardiac structure and function, sustained for 6 months, were comparable with syngeneic and allogeneic CDCs. Allogeneic CDCs stimulated endogenous regenerative mechanisms (cardiomyocyte cycling, recruitment of c-kit+ cells, angiogenesis) and increased myocardial VEGF, IGF-1 and HGF equally with syngeneic CDCs.
Allogeneic CDC transplantation without immunosuppression is safe, promotes cardiac regeneration and improves heart function in a rat myocardial infarction model, mainly through stimulation of endogenous repair mechanisms. This indirect mechanism of action rationalizes the persistence of benefit despite the evanescence of transplanted cell survival. This work motivates the testing of allogeneic human CDCs as a potential off-the-shelf product for cellular cardiomyoplasty.
PMCID: PMC3256094  PMID: 22086878
cardiac stem cells; allogeneic cell therapy; myocardial regeneration; paracrine effects
15.  Thioredoxin 1 Overexpression Extends Mainly the Earlier Part of Life Span in Mice 
We examined the effects of increased levels of thioredoxin 1 (Trx1) on resistance to oxidative stress and aging in transgenic mice overexpressing Trx1 [Tg(TRX1)+/0]. The Tg(TRX1)+/0 mice showed significantly higher Trx1 protein levels in all the tissues examined compared with the wild-type littermates. Oxidative damage to proteins and levels of lipid peroxidation were significantly lower in the livers of Tg(TRX1)+/0 mice compared with wild-type littermates. The survival study demonstrated that male Tg(TRX1)+/0 mice significantly extended the earlier part of life span compared with wild-type littermates, but no significant life extension was observed in females. Neither male nor female Tg(TRX1)+/0 mice showed changes in maximum life span. Our findings suggested that the increased levels of Trx1 in the Tg(TRX1)+/0 mice were correlated to increased resistance to oxidative stress, which could be beneficial in the earlier part of life span but not the maximum life span in the C57BL/6 mice.
PMCID: PMC3210956  PMID: 21873593
Thioredoxin; Transgenic mouse; Oxidative stress; Protein carbonylation; Aging
16.  Thioredoxin, oxidative stress, cancer and aging 
The Free Radical or Oxidative Stress Theory of Aging is one of the most popular theories in aging research and has been extensively studied over the past several decades. However, recent evidence using transgenic/knockout mice that overexpress or down-regulate antioxidant enzymes challenge the veracity of this theory since the animals show no increase or decrease in lifespan. These results seriously call into question the role of oxidative damage/stress in the aging process in mammals. Therefore, the theory requires significant modifications if we are to understand the relationship between aging and the regulation of oxidative stress. Our laboratory has been examining the impacts of thioredoxins (Trxs), in the cytosol and mitochondria, on aging and age-related diseases. Our data from mice that are either up-regulating or down-regulating Trx in different cellular compartments, that is, the cytosol or mitochondria, could shed some light on the role of oxidative stress and its pathophysiological effects. The results generated from our lab and others may indicate that: 1) changes in oxidative stress and the redox state in the cytosol, mitochondria or nucleus might play different roles in the aging process; 2) the role of oxidative stress and redox state could have different pathophysiological consequences in different tissues/cells, for example, mitotic vs. post-mitotic; 3) oxidative stress could have different pathophysiological impacts in young and old animals; and 4) the pathophysiological roles of oxidative stress and redox state could be controlled through changes in redox-sensitive signaling, which could have more diverse effects on pathophysiology than the accumulation of oxidative damage to various molecules. To critically test the role of oxidative stress on aging and age-related diseases, further study is required using animal models that regulate oxidative stress levels differently in each cellular compartment, each tissue/organ, and/or at different stages of life (young, middle and old) to change redox sensitive signaling pathways.
PMCID: PMC3886257  PMID: 24764510
Thioredoxin; Transgenic mouse; Knockout mouse; Oxidative stress; Cancer; aging
17.  Loss of manganese superoxide dismutase leads to abnormal growth and signal transduction in mouse embryonic fibroblasts 
Free radical biology & medicine  2010;49(8):1255-1262.
Manganese superoxide dismutase (MnSOD) in the mitochondria plays an important role in cellular defense against oxidative damage. Homozygous MnSOD knockout (Sod2−/−) mice are neonatal lethal, indicating the essential role of MnSOD in early development. To investigate the potential cellular abnormalities underlying the aborted development of Sod2−/− mice, we examined the growth of isolated mouse embryonic fibroblasts (MEF) from Sod2−/− mice. We found that the proliferation of Sod2−/− MEFs was significantly decreased when compared with wild type MEFs despite the absence of morphological differences. The Sod2−/− MEFs produced less cellular ATP, had lower O2 consumption, generated more superoxide, and expressed less Prdx3 protein. Furthermore, the loss of MnSOD dramatically altered several markers involved in cell proliferation and growth, including decreased growth stimulatory function of mTOR signaling and enhanced growth inhibitory function of GSK-3β signaling. Interestingly, the G protein coupled receptor-mediated intracellular Ca2+ ([Ca2+]i) signal transduction was also severely suppressed in Sod2−/− MEFs. Finally, the ratio of LC3-II/LC3-I, an index of autophagic activity, was increased in Sod2−/− MEFs, consistent with a reduction of mTOR signal transduction. These data demonstrate that MnSOD deficiency results in alterations in several key signaling pathways, which may contribute to the lethal phenotype of Sod2−/− mice.
PMCID: PMC3418666  PMID: 20638473
MnSOD; oxidative stress; ROS; signal transduction
18.  Short-Term Treatment With Rapamycin and Dietary Restriction Have Overlapping and Distinctive Effects in Young Mice 
Because rapamycin, an inhibitor of the nutrient sensor mammalian target of rapamycin, and dietary restriction both increase life span of mice, it has been hypothesized that they act through similar mechanisms. To test this hypothesis, we compared various biological parameters in dietary restriction mice (40% food restriction) and mice fed rapamycin (14 ppm). Both treatments led to a significant reduction in mammalian target of rapamycin signaling and a corresponding increase in autophagy. However, we observed striking differences in fat mass, insulin sensitivity, and expression of cell cycle and sirtuin genes in mice fed rapamycin compared with dietary restriction. Thus, although both treatments lead to significant downregulation of mammalian target of rapamycin signaling, these two manipulations have quite different effects on other physiological functions suggesting that they might increase life span through a common pathway as well as pathways that are altered differently by dietary restriction and rapamycin.
PMCID: PMC3598360  PMID: 22570137
Rapamycin; Dietary restriction; mTOR; Autophagy; Gene expression
19.  Expansion of human cardiac stem cells in physiological oxygen improves cell production efficiency and potency for myocardial repair 
Cardiovascular Research  2010;89(1):157-165.
The ex vivo expansion of cardiac stem cells from minimally invasive human heart biopsies yields tens of millions of cells within 3–4 weeks, but chromosomal abnormalities were frequently detected in preliminary production runs. Here we attempt to avoid aneuploidy and improve cell quality by expanding human cardiac stem cells in physiological low-oxygen (5% O2) conditions, rather than in traditional culture in a general CO2 incubator (20% O2).
Methods and results
Human heart biopsies (n = 16) were divided and processed in parallel to expand cardiac stem cells under 5% or 20% O2. Compared with 20% O2, 5% O2 culture doubled the cell production and markedly diminished the frequency of aneuploidy. Cells expanded in 5% O2 showed lower intracellular levels of reactive oxygen species, less cell senescence, and higher resistance to oxidative stress than those grown in 20% O2, although the expression of stem cell antigens and adhesion molecules was comparable between groups, as was the paracrine secretion of growth factors into conditioned media. In vivo, the implantation of 5% O2 cells into infarcted hearts of mice resulted in greater cell engraftment and better functional recovery than with conventionally cultured cells.
The expansion of human adult cardiac stem cells in low oxygen increased cell yield, and the resulting cells were superior by various key in vitro and in vivo metrics of cell quality. Physiological oxygen tensions in culture facilitate the ex vivo expansion of healthy, biologically potent stem cells.
PMCID: PMC3002866  PMID: 20675298
Physiological oxygen; Ex vivo expansion; Cardiac stem cells
20.  Isolation and expansion of functionally-competent cardiac progenitor cells directly from heart biopsies 
The adult heart contains reservoirs of progenitor cells that express embryonic and stem cell-related antigens. While these antigenically-purified cells are promising candidates for autologous cell therapy, clinical application is hampered by their limited abundance and tedious isolation methods. Methods that involve an intermediate cardiosphere-forming step have proven successful and are being tested clinically, but it is unclear whether the cardiosphere step is necessary. Accordingly, we investigated the molecular profile and functional benefit of cells that spontaneously emigrate from cardiac tissue in primary culture. Adult Wistar-Kyoto rat hearts were minced, digested and cultured as separate anatomical regions. Loosely-adherent cells that surround the plated tissue were harvested weekly for a total of five harvests. Genetic lineage tracing demonstrated that a small proportion of the direct outgrowth from cardiac samples originates from myocardial cells. This outgrowth contains sub-populations of cells expressing embryonic (SSEA-1) and stem cell-related antigens (c-Kit, abcg2) that varied with time in culture but not with the cardiac chamber of origin. This direct outgrowth, and its expanded progeny, underwent marked in vitro angiogenic/cardiogenic differentiation and cytokine secretion (IGF-1, VGEF). In vivo effects included long-term functional benefits as gauged by MRI following cell injection in a rat model of myocardial infarction. Outgrowth cells afforded equivalent functional benefits to cardiosphere-derived cells, which require more processing steps to manufacture. These results provide the basis for a simplified and efficient process to generate autologous cardiac progenitor cells (and mesenchymal supporting cells) to augment clinically-relevant approaches for myocardial repair.
PMCID: PMC2885498  PMID: 20211627
cell therapy; myocardial infarction; cardiac regeneration; angiogenesis; ventricular remodelling
21.  Magnetic Targeting Enhances Engraftment and Functional Benefit of Iron-Labeled Cardiosphere-Derived Cells in Myocardial Infarction 
Circulation research  2010;106(10):1570-1581.
The success of cardiac stem cell therapies is limited by low cell retention, due at least in part to washout via coronary veins.
We sought to counter the efflux of transplanted cells by rendering them magnetically-responsive and imposing an external magnetic field on the heart during and immediately after injection.
Methods and Results
Cardiosphere-derived cells (CDCs) were labeled with superparamagnetic microspheres (SPMs). In vitro studies revealed that cell viability and function were minimally affected by SPM labeling. SPM-labeled rat CDCs were injected intramyocardially, with and without a superimposed magnet. With magnetic targeting, cells were visibly attracted towards the magnet and accumulated around the ischemic zone. In contrast, the majority of non-targeted cells washed out immediately after injection. Fluorescence imaging revealed more retention of transplanted cells in the heart, and less migration into other organs, in the magnetically-targeted group. Quantitative PCR confirmed that magnetic targeting enhanced cell retention (at 24 hours) and engraftment (at 3 weeks) in the recipient hearts by ∼3-fold compared to non-targeted cells. Morphometric analysis revealed maximal attenuation of LV remodeling, and echocardiography showed the greatest functional improvement, in the magnetic targeting group. Histologically, more engrafted cells were evident with magnetic targeting, but there was no incremental inflammation.
Magnetic targeting enhances cell retention, engraftment and functional benefit. This novel method to improve cell therapy outcomes offers the potential for rapid translation into clinical applications.
PMCID: PMC2885138  PMID: 20378859
cardiac progenitor cells; cell transplantation; myocardial infarction; targeted cell delivery
22.  Analysis of synonymous codon usage in Hepatitis A virus 
Virology Journal  2011;8:174.
Hepatitis A virus is the causative agent of type A viral hepatitis, which causes occasional acute hepatitis. Nevertheless, little information about synonymous codon usage pattern of HAV genome in the process of its evolution is available. In this study, the key genetic determinants of codon usage in HAV were examined.
The overall extent of codon usage bias in HAV is high in Picornaviridae. And the patterns of synonymous codon usage are quite different in HAV genomes from different location. The base composition is closely correlated with codon usage bias. Furthermore, the most important determinant that results in such a high codon bias in HAV is mutation pressure rather than natural selection.
HAV presents a higher codon usage bias than other members of Picornaviridae. Compositional constraint is a significant element that influences the variation of synonymous codon usage in HAV genome. Besides, mutation pressure is supposed to be the major factor shaping the hyperendemic codon usage pattern of HAV.
PMCID: PMC3087699  PMID: 21496278
23.  Mice Deficient in Both Mn Superoxide Dismutase and Glutathione Peroxidase-1 Have Increased Oxidative Damage and a Greater Incidence of Pathology but No Reduction in Longevity 
To test the impact of increased mitochondrial oxidative stress as a mechanism underlying aging and age-related pathologies, we generated mice with a combined deficiency in two mitochondrial-localized antioxidant enzymes, Mn superoxide dismutase (MnSOD) and glutathione peroxidase-1 (Gpx-1). We compared life span, pathology, and oxidative damage in Gpx1−/−, Sod2+/−Gpx1+/−, Sod2+/−Gpx1−/−, and wild-type control mice. Oxidative damage was elevated in Sod2+/−Gpx1−/− mice, as shown by increased DNA oxidation in liver and skeletal muscle and increased protein oxidation in brain. Surprisingly, Sod2+/−Gpx1−/− mice showed no reduction in life span, despite increased levels of oxidative damage. Consistent with the important role for oxidative stress in tumorigenesis during aging, the incidence of neoplasms was significantly increased in the older Sod2+/−Gpx1−/− mice (28–30 months). Thus, these data do not support a significant role for increased oxidative stress as a result of compromised mitochondrial antioxidant defenses in modulating life span in mice and do not support the oxidative stress theory of aging.
PMCID: PMC2781787  PMID: 19776219
Oxidative stress; Longevity
24.  Dedifferentiation and Proliferation of Mammalian Cardiomyocytes 
PLoS ONE  2010;5(9):e12559.
It has long been thought that mammalian cardiomyocytes are terminally-differentiated and unable to proliferate. However, myocytes in more primitive animals such as zebrafish are able to dedifferentiate and proliferate to regenerate amputated cardiac muscle.
Methodology/Principal Findings
Here we test the hypothesis that mature mammalian cardiomyocytes retain substantial cellular plasticity, including the ability to dedifferentiate, proliferate, and acquire progenitor cell phenotypes. Two complementary methods were used: 1) cardiomyocyte purification from rat hearts, and 2) genetic fate mapping in cardiac explants from bi-transgenic mice. Cardiomyocytes isolated from rodent hearts were purified by multiple centrifugation and Percoll gradient separation steps, and the purity verified by immunostaining and RT-PCR. Within days in culture, purified cardiomyocytes lost their characteristic electrophysiological properties and striations, flattened and began to divide, as confirmed by proliferation markers and BrdU incorporation. Many dedifferentiated cardiomyocytes went on to express the stem cell antigen c-kit, and the early cardiac transcription factors GATA4 and Nkx2.5. Underlying these changes, inhibitory cell cycle molecules were suppressed in myocyte-derived cells (MDCs), while microRNAs known to orchestrate proliferation and pluripotency increased dramatically. Some, but not all, MDCs self-organized into spheres and re-differentiated into myocytes and endothelial cells in vitro. Cell fate tracking of cardiomyocytes from 4-OH-Tamoxifen-treated double-transgenic MerCreMer/ZEG mouse hearts revealed that green fluorescent protein (GFP) continues to be expressed in dedifferentiated cardiomyocytes, two-thirds of which were also c-kit+.
Contradicting the prevailing view that they are terminally-differentiated, postnatal mammalian cardiomyocytes are instead capable of substantial plasticity. Dedifferentiation of myocytes facilitates proliferation and confers a degree of stemness, including the expression of c-kit and the capacity for multipotency.
PMCID: PMC2933247  PMID: 20838637
25.  Validation of the Cardiosphere Method to Culture Cardiac Progenitor Cells from Myocardial Tissue 
PLoS ONE  2009;4(9):e7195.
At least four laboratories have shown that endogenous cardiac progenitor cells (CPCs) can be grown directly from adult heart tissue in primary culture, as cardiospheres or their progeny (cardiosphere-derived cells, CDCs). Indeed, CDCs are already being tested in a clinical trial for cardiac regeneration. Nevertheless, the validity of the cardiosphere strategy to generate CPCs has been called into question by reports based on variant methods. In those reports, cardiospheres are argued to be cardiomyogenic only because of retained cardiomyocytes, and stem cell activity has been proposed to reflect hematological contamination. We use a variety of approaches (including genetic lineage tracing) to show that neither artifact is applicable to cardiospheres and CDCs grown using established methods, and we further document the stem cell characteristics (namely, clonogenicity and multilineage potential) of CDCs.
Methodology/Principal Findings
CPCs were expanded from human endomyocardial biopsies (n = 160), adult bi-transgenic MerCreMer-Z/EG mice (n = 6), adult C57BL/6 mice (n = 18), adult GFP+ C57BL/6 transgenic mice (n = 3), Yucatan mini pigs (n = 67), adult SCID beige mice (n = 8), and adult Wistar-Kyoto rats (n = 80). Cellular yield was enhanced by collagenase digestion and process standardization; yield was reduced in altered media and in specific animal strains. Heparinization/retrograde organ perfusion did not alter the ability to generate outgrowth from myocardial sample. The initial outgrowth from myocardial samples was enriched for sub-populations of CPCs (c-Kit+), endothelial cells (CD31+, CD34+), and mesenchymal cells (CD90+). Lineage tracing using MerCreMer-Z/EG transgenic mice revealed that the presence of cardiomyocytes in the cellular outgrowth is not required for the generation of CPCs. Rat CDCs are shown to be clonogenic, and cloned CDCs exhibit spontaneous multineage potential.
This study demonstrates that direct culture and expansion of CPCs from myocardial tissue is simple, straightforward, and reproducible when appropriate techniques are used.
PMCID: PMC2745677  PMID: 19779618

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