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1.  Overexpression of myocardin induces partial transdifferentiation of human‐induced pluripotent stem cell‐derived mesenchymal stem cells into cardiomyocytes 
Physiological Reports  2014;2(2):e00237.
Abstract
Mesenchymal stem cells (MSCs) derived from human‐induced pluripotent stem cells (iPSCs) show superior proliferative capacity and therapeutic potential than those derived from bone marrow (BM). Ectopic expression of myocardin further improved the therapeutic potential of BM‐MSCs in a mouse model of myocardial infarction. The aim was of this study was to assess whether forced myocardin expression in iPSC‐MSCs could further enhance their transdifferentiation to cardiomyocytes and improve their electrophysiological properties for cardiac regeneration. Myocardin was overexpressed in iPSC‐MSCs using viral vectors (adenovirus or lentivirus). The expression of smooth muscle cell and cardiomyocyte markers, and ion channel genes was examined by reverse transcription‐polymerase chain reaction (RT‐PCR), immunofluorescence staining and patch clamp. The conduction velocity of the neonatal rat ventricular cardiomyocytes cocultured with iPSC‐MSC monolayer was measured by multielectrode arrays recording plate. Myocardin induced the expression of α‐MHC, GATA4, α‐actinin, cardiac MHC, MYH11, calponin, and SM α‐actin, but not cTnT, β‐MHC, and MLC2v in iPSC‐MSCs. Overexpression of myocardin in iPSC‐MSC enhanced the expression of SCN9A and CACNA1C, but reduced that of KCa3.1 and Kir2.2 in iPSC‐MSCs. Moreover, BKCa, IKir, ICl, Ito and INa.TTX were detected in iPSC‐MSC with myocardin overexpression; while only BKCa, IKir, ICl, IKDR, and IKCa were noted in iPSC‐MSC transfected with green florescence protein. Furthermore, the conduction velocity of iPSC‐MSC was significantly increased after myocardin overexpression. Overexpression of myocardin in iPSC‐MSCs resulted in partial transdifferentiation into cardiomyocytes phenotype and improved the electrical conduction during integration with mature cardiomyocytes.
Forced myocardin expression in human‐induced pluripotent stem cell (hiPSC)‐derived mesenchymal stem cells lead to partial transdifferentiation into cardiomyocytes and smooth muscle cells phenotypes through modification in ion channel expression profile and electrical conduction velocity.
doi:10.1002/phy2.237
PMCID: PMC3966242  PMID: 24744906
Cardiomyocytes; mesenchymal stem cell; myocardin; transdifferentiation
2.  Sudden Cardiac Death After Myocardial Infarction in Type 2 Diabetic Patients With No Residual Myocardial Ischemia 
Diabetes Care  2012;35(12):2564-2569.
OBJECTIVE
Diabetes mellitus (DM) is a well-established risk factor for coronary artery disease. Nonetheless, it remains unclear whether DM contributes to sudden cardiac death in patients who survive myocardial infarction (MI). The objective of this study was to compare the incidence of sudden cardiac death post-MI in diabetic and nondiabetic patients with no residual myocardial ischemia.
RESEARCH DESIGN AND METHODS
A total of 610 consecutive post-MI patients referred to a cardiac rehabilitation program with negative exercise stress test were studied.
RESULTS
Of these, 236 patients had DM at baseline. Over a mean follow-up of 5 years, 67 patients with DM (28.4%) and 76 of 374 patients without DM (20.2%) had died with a hazard ratio (HR) of 1.74 (95% CI: 1.28–2.56; P < 0.001). Patients with DM also had a higher incidence of cardiac death (1.84 [1.16–3.21]; P = 0.01), principally due to a higher incidence of sudden cardiac death (2.14 [1.22–4.23]; P < 0.001). Multiple Cox regression analysis revealed that only DM (adjusted HR: 1.9 [95% CI: 1.04–3.40]; P = 0.04), left ventricular ejection fraction (LVEF) ≤30% (3.6 [1.46–8.75]; P < 0.01), and New York Heart Association functional class >II (4.2 [1.87–9.45]; P < 0.01) were independent predictors for sudden cardiac death. Among patients with DM, the 5-year sudden cardiac death rate did not differ significantly among those with LVEF ≤30%, LVEF 31–50%, or LVEF >50% (8.8 vs. 7.8 vs. 6.8%, respectively; P = 0.83).
CONCLUSIONS
Post-MI patients with DM, even in the absence of residual myocardial ischemia clinically, were at higher risk of sudden cardiac death than their non-DM counterparts.
doi:10.2337/dc12-0118
PMCID: PMC3507604  PMID: 22875229
3.  Nutrient supplemented serum-free medium increases cardiomyogenesis efficiency of human pluripotent stem cells 
World Journal of Stem Cells  2013;5(3):86-97.
AIM: To development of an improved p38 MAPK inhibitor-based serum-free medium for embryoid body cardiomyocyte differentiation of human pluripotent stem cells.
METHODS: Human embryonic stem cells (hESC) differentiated to cardiomyocytes (CM) using a p38 MAPK inhibitor (SB203580) based serum-free medium (SB media). Nutrient supplements known to increase cell viability were added to SB medium. The ability of these supplements to improve cardiomyogenesis was evaluated by measurements of cell viability, total cell count, and the expression of cardiac markers via flow cytometry. An improved medium containing Soy hydrolysate (HySoy) and bovine serum albumin (BSA) (SupSB media) was developed and tested on 2 additional cell lines (H1 and Siu-hiPSC). Characterization of the cardiomyocytes was done by immunohistochemistry, electrophysiology and quantitative real-time reverse transcription-polymerase chain reaction.
RESULTS: hESC cell line, HES-3, differentiating in SB medium for 16 d resulted in a cardiomyocyte yield of 0.07 ± 0.03 CM/hESC. A new medium (SupSB media) was developed with the addition of HySoy and BSA to SB medium. This medium resulted in 2.6 fold increase in cardiomyocyte yield (0.21 ± 0.08 CM/hESC). The robustness of SupSB medium was further demonstrated using two additional pluripotent cell lines (H1, hESC and Siu1, hiPSC), showing a 15 and 9 fold increase in cardiomyocyte yield respectively. The age (passage number) of the pluripotent cells did not affect the cardiomyocyte yields. Embryoid body (EB) cardiomyocytes formed in SupSB medium expressed canonical cardiac markers (sarcomeric α-actinin, myosin heavy chain and troponin-T) and demonstrated all three major phenotypes: nodal-, atrial- and ventricular-like. Electrophysiological characteristics (maximum diastolic potentials and action potential durations) of cardiomyocytes derived from SB and SupSB media were similar.
CONCLUSION: The nutrient supplementation (HySoy and BSA) leads to increase in cell viability, cell yield and cardiac marker expression during cardiomyocyte differentiation, translating to an overall increase in cardiomyocyte yield.
doi:10.4252/wjsc.v5.i3.86
PMCID: PMC3723792  PMID: 23904910
Soy hydrolysate; Bovine serum albumin; Differentiation; Cardiomyocyte; Human embryonic stem cells; Human induced pluripotent stem cells
4.  Modeling of lamin A/C mutation premature cardiac aging using patient-specific induced pluripotent stem cells 
Aging (Albany NY)  2012;4(11):803-822.
AIMS
We identified an autosomal dominant non-sense mutation (R225X) in exon 4 of the lamin A/C (LMNA) gene in a Chinese family spanning 3 generations with familial dilated cardiomyopathy (DCM). In present study, we aim to generate induced pluripotent stem cells derived cardiomyocytes (iPSC-CMs) from an affected patient with R225X and another patient bearing LMNA frame-shift mutation for drug screening.
METHODS and RESULTS
Higher prevalence of nuclear bleb formation and micronucleation was present in LMNAR225X/WT and LMNAFramshift/WT iPSC-CMs. Under field electrical stimulation, percentage of LMNA-mutated iPSC-CMs exhibiting nuclear senescence and cellular apoptosis markedly increased. shRNA knockdown of LMNA replicated those phenotypes of the mutated LMNA field electrical stress. Pharmacological blockade of ERK1/2 pathway with MEK1/2 inhibitors, U0126 and selumetinib (AZD6244) significantly attenuated the pro-apoptotic effects of field electric stimulation on the mutated LMNA iPSC-CMs.
CONCLUSION
LMNA-related DCM was modeled in-vitro using patient-specific iPSC-CMs. Our results demonstrated that haploinsufficiency due to R225X LMNA non-sense mutation was associated with accelerated nuclear senescence and apoptosis of iPSC- CMs under electrical stimulation, which can be significantly attenuated by therapeutic blockade of stress-related ERK1/2 pathway.
PMCID: PMC3560431  PMID: 23362510
Dilated cardiomyopathy; induced pluripotent stem cells; LMNA
5.  Generation of induced pluripotent stem cell lines from 3 distinct laminopathies bearing heterogeneous mutations in lamin A/C 
Aging (Albany NY)  2011;3(4):380-390.
The term laminopathies defines a group of genetic disorders caused by defects in the nuclear envelope, mostly the lamins. Lamins are the main constituents of the nuclear lamina, a filamentous meshwork associated with the inner nuclear membrane that provides mechanical stability and plays important roles in processes such as transcription, DNA replication and chromatin organization. More than 300 mutations in lamin A/C have been associated with diverse clinical phenotypes, understanding the molecular basis of these diseases may provide a rationale for treating them. Here we describe the generation of induced pluripotent stem cells (iPSCs) from a patient with inherited dilated cardiomiopathy and 2 patients with distinct accelerated forms of aging, atypical Werner syndrome and Hutchinson Gilford progeria, all of which are caused by mutations in lamin A/C. These cell lines were pluripotent and displayed normal nuclear membrane morphology compared to donor fibroblasts. Their differentiated progeny reproduced the disease phenotype, reinforcing the idea that they represent excellent tools for understanding the role of lamin A/C in normal physiology and the clinical diversity associated with these diseases.
PMCID: PMC3117453  PMID: 21483033
reprogramming; induced pluripotent stem cells; lamin A/C; dilated cardiomyopathy; atypical Werner syndrome; Hutchinson Gilford progeria
6.  Synergistic effects of Inward Rectifier (IK1) and Pacemaker (If) Currents on the Induction of Bioengineered Cardiac Automaticity 
Introduction
Normal heart rhythms originate in the sinoatrial node. HCN-encoded funny current (If) and the Kir2-encoded inward rectifier (IK1) counteract each other by respectively oscillating and stabilizing the negative resting membrane potential, controlling action potential firing. Therefore, IK1 suppression and If overexpression have been independently exploited to convert cardiomyocytes (CMs) into AP-firing bioartificial pacemakers. Although the two strategies have been largely assumed synergistic, their complementarity has not been investigated.
Methods and Results
We explored the inter-relationships of automaticity, If and IK1 by transducing single left ventricular (LV) CMs isolated from guinea pig hearts with the recombinant adenoviruses Ad-CMV-GFP-IRES-HCN1-ΔΔΔ and/or Ad-CGI-Kir2.1 to mediate their current densities via whole-cell patch clamp technique at 37°C. Results showed that Ad-CGI-HCN1-ΔΔΔ- but not Ad-CGI-Kir2.1-transduction induced automaticity (181.1±13.1 bpm). Interestingly, Ad-CGI-HCN1-ΔΔΔ/Ad-CGI-Kir2.1 cotransduction significantly promoted the induced firing frequency (320.0±15.8 bpm; p<0.05). Correlation analysis revealed that the firing frequency, phase 4 slope and APD90 of AP-firing LV CMs were correlated to If (R2>0.7) only when -2>IK1>-4 pA/pF but not to IK1 over the entire If ranges examined (0.02If>-4 pA/pF. As anticipated, however, APD90 was correlated to IK1 (R2=0.4).
Conclusion
We conclude that an optimal level of IK1 maintains a voltage range for If to operate most effectively during a dynamic cardiac cycle.
doi:10.1111/j.1540-8167.2009.01475.x
PMCID: PMC2739246  PMID: 19460073
If; IK1; bioartificial pacemaker; automaticity; synergism
7.  Incidence, clinical characteristics and outcome of congestive heart failure as the initial presentation in patients with primary hyperthyroidism 
Heart  2006;93(4):483-487.
Background
There are limited systematic data on the incidence, clinical characteristics and outcomes of congestive heart failure (CHF) in patients with hyperthyroidism. The aim of this study was to investigate the incidence, clinical characteristics and outcome of CHF as the initial presentation in patients with primary hyperthyroidism.
Methods
The prevalence, clinical characteristics and outcome of CHF was studied in 591 consecutive patients (mean (SD) age 45 (1) years, 140 men) who presented with primary hyperthyroidism.
Results
CHF was the presenting condition in 34 patients (5.8%) with hyperthyroidism. The presence of atrial fibrillation at presentation (OR 37.4, 95% CI 9.72 to 144.0, p<0.001) was an independent predictor for the occurrence of CHF. Of the 34 patients with CHF, 16 (47%) had systolic left ventricular dysfunction with left ventricular ejection fraction (LVEF)<50%. They were predominantly male (OR 26.6, 95% CI 2.6 to 272.5, p = 0.006) and had a lower serum thyroxine level (OR 0.93, 95% CI 0.87 to 0.99, p = 0.044) than patients with preserved left ventricular systolic function. In these patients, LVEF (55 (4)% vs 30 (2)%, p<0.001) and New York Heart Association functional class (1.2 (0.1) vs 2.5 (0.2), p<0.001) improved significantly 3 months after achieving euthyroid status. Systolic left ventricular dysfunction (mean (SD) LVEF 38 (4)%) persisted on long‐term follow‐up in five patients: no clinical parameter could be identified to predict the occurrence of this persistent cardiomyopathy (p>0.05).
Conclusion
CHF was the initial clinical presentation in approximately 6% of patients with hyperthyroidism, and half of them had left ventricular systolic dysfunction. Symptoms of CHF subsided and LVEF improved after treatment for hyperthyroidism. Nonetheless, one‐third of these patients developed persistent dilated cardiomyopathy.
doi:10.1136/hrt.2006.100628
PMCID: PMC1861478  PMID: 17005710
8.  Incidence and predictors of upper gastrointestinal bleeding in patients receiving low-dose aspirin for secondary prevention of cardiovascular events in patients with coronary artery disease 
AIM: The use of low-dose aspirin to prevent cardiovascular disease events is well established. However, the incidence and predictors of upper gastrointestinal bleeding (UGIB) with its use are unknown. We studied prospectively the incidence and outcome of peptic ulceration in low-dose aspirin users.
METHODS: A total of 991 patients with coronary artery disease (CAD) on low-dose aspirin were prospectively followed-up for two years for the occurrence and clinical features of first hospitalized episode of UGIB.
RESULTS: UGIB had a bimodal presentation with 45% occurring within four months of aspirin initiation and had an overall prevalence of 1.5% per year. There was no UGIB-related death. Hypertension (OR = 4.6, 95%CI 1.5 - 14.7, P = 0.009), history of peptic ulceration (OR = 3.1, 95%CI 1.1 - 9.0, P = 0.039), tertiary education (OR = 3.08, 95%CI 1.1 - 9.0, P = 0.039) and higher lean body mass (P = 0.016) were independent factors associated with UGIB. Use of nitrate did not reduce UGIB.
CONCLUSION: The incidence of UGIB in patients with CAD on long-term low-dose aspirin is low, but is accompanied with significant morbidity. With prolonged use of aspirin, UGIB continues to be a problem for those with risk factors and especially in patients with a history of peptic ulcers, in which UGIB tends to occur early after aspirin therapy.
doi:10.3748/wjg.v12.i18.2923
PMCID: PMC4087812  PMID: 16718820
Coronary artery disease; Aspirin; Gastrointestinal bleeding
9.  Incremental prognostic value of global longitudinal strain in patients with type 2 diabetes mellitus 
Background
Patients with type 2 diabetes mellitus (T2DM) are at high risk for cardiovascular events. The aim of the study was to assess whether global longitudinal strain (GLS) provides prognostic value in these patients.
Methods
A total of 247 T2DM patients without history of cardiovascular complications and participated in the CDATS study were prospectively enrolled. Left ventricular (LV) systolic function was assessed by LV ejection fraction and speckle tracking derived LV systolic GLS. Diastolic function was assessed by E/E′ ratio defined as the passive trans-mitral LV inflow velocity to tissue Doppler imaging velocity of the medial mitral annulus. Cardiovascular event included acute coronary syndrome, cerebrovascular stroke, hospitalization for heart failure and cardiovascular death.
Results
A total of 18 cardiovascular events occurred during a median follow-up duration of 33 months. Both E/E′ ratio [hazard ratio (HR) 1.15, P < 0.01] and GLS (HR 1.39, P < 0.01) were independently associated with cardiovascular events. Importantly, GLS provided incremental prognostic information in addition to clinical data, HbA1c and E/E′ ratio (Chi square 77.46, P = 0.04). Receiver-operator characteristic curve analysis demonstrated that E/E′ ratio [area under curve (AUC) 0.66, P = 0.03] and GLS (AUC 0.72, P < 0.01) were strong predictors of cardiovascular events. Kaplan–Meier analysis showed that patients with E/E′ > 13.6 or GLS > −17.9 % were associated with cardiovascular events. The presence of either a high E/E′ ratio or an impaired GLS provides an excellent negative predictive value of cardiovascular events in these patients.
Conclusions
In T2DM patients with no history of cardiovascular disease, impaired GLS was associated with cardiovascular events and provided incremental prognostic value.
doi:10.1186/s12933-016-0333-5
PMCID: PMC4738770  PMID: 26842466
Type 2 diabetes mellitus; Left ventricular function; Cardiovascular events
10.  Exome-wide association analysis reveals novel coding sequence variants associated with lipid traits in Chinese 
Nature Communications  2015;6:10206.
Blood lipids are important risk factors for coronary artery disease (CAD). Here we perform an exome-wide association study by genotyping 12,685 Chinese, using a custom Illumina HumanExome BeadChip, to identify additional loci influencing lipid levels. Single-variant association analysis on 65,671 single nucleotide polymorphisms reveals 19 loci associated with lipids at exome-wide significance (P<2.69 × 10−7), including three Asian-specific coding variants in known genes (CETP p.Asp459Gly, PCSK9 p.Arg93Cys and LDLR p.Arg257Trp). Furthermore, missense variants at two novel loci—PNPLA3 p.Ile148Met and PKD1L3 p.Thr429Ser—also influence levels of triglycerides and low-density lipoprotein cholesterol, respectively. Another novel gene, TEAD2, is found to be associated with high-density lipoprotein cholesterol through gene-based association analysis. Most of these newly identified coding variants show suggestive association (P<0.05) with CAD. These findings demonstrate that exome-wide genotyping on samples of non-European ancestry can identify additional population-specific possible causal variants, shedding light on novel lipid biology and CAD.
An important risk factor for coronary artery disease is the level of blood lipids. Here the authors conduct an exome-wide association study in Chinese cohorts and identify three novel loci associated with lipid levels as well as three Asian-specific variants in known loci.
doi:10.1038/ncomms10206
PMCID: PMC4703860  PMID: 26690388
11.  The p53-induced lincRNA-p21 derails somatic cell reprogramming by sustaining H3K9me3 and CpG methylation at pluripotency gene promoters 
Cell Research  2014;25(1):80-92.
Recent studies have boosted our understanding of long noncoding RNAs (lncRNAs) in numerous biological processes, but few have examined their roles in somatic cell reprogramming. Through expression profiling and functional screening, we have identified that the large intergenic noncoding RNA p21 (lincRNA-p21) impairs reprogramming. Notably, lincRNA-p21 is induced by p53 but does not promote apoptosis or cell senescence in reprogramming. Instead, lincRNA-p21 associates with the H3K9 methyltransferase SETDB1 and the maintenance DNA methyltransferase DNMT1, which is facilitated by the RNA-binding protein HNRNPK. Consequently, lincRNA-p21 prevents reprogramming by sustaining H3K9me3 and/or CpG methylation at pluripotency gene promoters. Our results provide insight into the role of lncRNAs in reprogramming and establish a novel link between p53 and heterochromatin regulation.
doi:10.1038/cr.2014.165
PMCID: PMC4650593  PMID: 25512341
somatic cell reprogramming; long noncoding RNAs; p53; lincRNA-p21; heterochromatin; H3K9 methylation; DNA methylation
12.  Altered myocardial response in patients with diabetic retinopathy: an exercise echocardiography study 
Background
Type 2 diabetes mellitus (T2DM) complicated by retinopathy is associated with altered left ventricular (LV) structure and resting myocardial dysfunction unlike T2DM without retinopathy. The myocardial response to stress has not been compared in patients with and without diabetic retinopathy. The aim of this retrospective study was to determine the relationship between retinopathy and myocardial function in patients with T2DM at rest and during exercise echocardiography.
Methods
134 patients with T2DM and no evidence of underlying coronary artery disease were recruited. All patients underwent retinal photography to screen for diabetic retinopathy, and resting and exercise echocardiography. Resting echocardiography was analyzed by conventional echocardiographic parameters and speckle tracking derived global longitudinal strain (GLS). Exercise echocardiography parameters included diastolic function reserve index (DFRI) and stress GLS.
Results
The mean age of participants was 60 years and 49 % were male. Diabetic retinopathy was identified in 43 patients (32 %). Resting echocardiography revealed that those with diabetic retinopathy had a higher prevalence of impaired diastolic function, higher E/E′ ratio (LV filling pressures) and impaired resting GLS compared with those without. Exercise echocardiography revealed that those with diabetic retinopathy also had more impaired DFRI and stress GLS. Multivariable analysis showed that the presence of diabetic retinopathy was independently associated with high resting E/E′, diastolic dysfunction grade, impaired resting GLS, low DFRI and impaired stress GLS.
Conclusions
In conclusion, the presence of diabetic retinopathy was independently associated with impaired resting myocardial function (diastolic and systolic function) and myocardial function during stress (evaluated by DFRI and stress GLS).
Electronic supplementary material
The online version of this article (doi:10.1186/s12933-015-0281-5) contains supplementary material, which is available to authorized users.
doi:10.1186/s12933-015-0281-5
PMCID: PMC4574544  PMID: 26382215
Diabetic cardiomyopathy; Cardiac dysfunction; Exercise echocardiography; Diabetic retinopathy; Cardiac function reserve
13.  Duration of dual antiplatelet therapy after drug-eluting stent implantation: Meta-analysis of large randomised controlled trials 
Scientific Reports  2015;5:13204.
Patients receive dual antiplatelet therapy (DAPT) for 6–12 months after drug-eluting stents (DES) implantation. The efficacy and safety of prolonged DAPT has been questioned. Therefore, we performed a meta-analysis on randomised trials comparing different DAPT durations. Literature was searched on trials comparing different DAPT durations. For inclusion, reports must report frequency of cardiovascular and bleeding events. Ten trials were included. Compared to 12 months, DAPT beyond 12 months was associated with fewer myocardial infarctions (OR 0.58 95%CI: 0.40–0.84) and stent thrombosis (OR 0.35 95%CI: 0.20–0.62), but more major bleeds (OR 1.60 95%CI: 1.22–2.11) and all-cause (OR 1.30 95%CI: 1.02–1.66) mortality. There was no significant alteration in risk of stroke (OR 0.93 95%CI: 0.66–1.31) or cardiac (OR 1.12 95%CI: 0.73–1.71) mortality. Compared to less than 12 months DAPT, 12 months DAPT did not reduce risk of myocardial infarction, stent thrombosis, strokes, cardiac or all-cause mortality, but increased the risk of major bleeds (OR 1.60 95%CI: 1.22–2.11). DAPT beyond 12 months reduce risk of myocardial infarction and stent thrombosis, but there is substantial increase in major bleeding risk and all-cause mortality which need to be addressed. DAPT beyond 12 months does not appear to alter the risk of stroke.
doi:10.1038/srep13204
PMCID: PMC4538605  PMID: 26278959
14.  Plant Natural Products Calycosin and Gallic Acid Synergistically Attenuate Neutrophil Infiltration and Subsequent Injury in Isoproterenol-Induced Myocardial Infarction: A Possible Role for Leukotriene B4 12-Hydroxydehydrogenase? 
Leukotriene B4 12-hydroxydehydrogenase (LTB4DH) catalyzes the oxidation of proinflammatory LTB4 into less bioactive 12-oxo-LTB4. We recently discovered that LTB4DH was induced by two different natural products in combination. We previously isolated gallic acid from Radix Paeoniae through a bioactivity-guided fractionation procedure. The purpose of this study is to test the hypothesis that LTB4DH inducers may suppress neutrophil-mediated inflammation in myocardial infarction. We first isolated the active compound(s) from another plant, Radix Astragali, by the similar strategy. By evaluating LTB4DH induction, we identified calycosin and formononetin from Radix Astragali by HPLC-ESI-MS technique. We confirmed that gallic acid and commercial calycosin or formononetin could synergistically induce LTB4DH expression in HepG2 cells and human neutrophils. Moreover, calycosin and gallic acid attenuated the effects of LTB4 on the survival and chemotaxis of neutrophil cell culture. We further demonstrated that calycosin and gallic acid synergistically suppressed neutrophil infiltration and protected cardiac integrity in the isoproterenol-induced mice model of myocardial infarction. Calycosin and gallic acid dramatically suppressed isoproterenol-induced increase in myeloperoxidase (MPO) activity and malondialdehyde (MDA) level. Collectively, our results suggest that LTB4DH inducers (i.e., calycosin and gallic acid) may be a novel combined therapy for the treatment of neutrophil-mediated myocardial injury.
doi:10.1155/2015/434052
PMCID: PMC4523677  PMID: 26265982
15.  Human Ocular Epithelial Cells Endogenously Expressing SOX2 and OCT4 Yield High Efficiency of Pluripotency Reprogramming 
PLoS ONE  2015;10(7):e0131288.
A variety of pluripotency reprogramming frequencies from different somatic cells has been observed, indicating cell origin is a critical contributor for efficiency of pluripotency reprogramming. Identifying the cell sources for efficient induced pluripotent stem cells (iPSCs) generation, and defining its advantages or disadvantages on reprogramming, is therefore important. Human ocular tissue-derived conjunctival epithelial cells (OECs) exhibited endogenous expression of reprogramming factors OCT4A (the specific OCT 4 isoform on pluripotency reprogramming) and SOX2. We therefore determined whether OECs could be used for high efficiency of iPSCs generation. We compared the endogenous expression levels of four pluripotency factors and the pluripotency reprograming efficiency of human OECs with that of ocular stromal cells (OSCs). Real-time PCR, microarray analysis, Western blotting and immunostaining assays were employed to compare OECiPSCs with OSCiPSCs on molecular bases of reprogramming efficiency and preferred lineage-differentiation potential. Using the traditional KMOS (KLF4, C-MYC, OCT4 and SOX2) reprogramming protocol, we confirmed that OECs, endogenously expressing reprogramming factors OCT4A and SOX2, yield very high efficiency of iPSCs generation (~1.5%). Furthermore, higher efficiency of retinal pigmented epithelial differentiation (RPE cells) was observed in OECiPSCs compared to OSCiPSCs or skin fibroblast iMR90iPSCs. The findings in this study suggest that conjunctival-derived epithelial (OECs) cells can be easier converted to iPSCs than conjunctival-derived stromal cells (OSCs). This cell type may also have advantages in retinal pigmented epithelial differentiation.
doi:10.1371/journal.pone.0131288
PMCID: PMC4489496  PMID: 26131692
16.  Impact of Antithrombotic Therapy in Atrial Fibrillation on the Presentation of Coronary Artery Disease 
PLoS ONE  2015;10(6):e0131479.
Background
Little is known about whether atrial fibrillation is a presentation of coronary disease. There is a paucity of knowledge about their causal relationship and also the impact of different antithrombotic strategies on the subsequent presentation of symptomatic coronary disease.
Methods and Results
We studied 7,526 Chinese patients diagnosed with non-valvular atrial fibrillation and no documented history of coronary artery disease. The primary endpoint was the new occurrence of coronary artery disease—either stable coronary artery disease or acute coronary syndrome. After a mean follow-up of 3.2±3.5 years (24,071 patient-years), a primary endpoint occurred in 987 patients (13.1%). The overall annual incidence of coronary artery disease was 4.10%/year. No significant differences in age, sex, and mean CHA2DS2-VASc score were observed between patients with and without the primary endpoint. When stratified according to the antithrombotic strategies applied for stroke prevention, the annual incidence of coronary artery disease was 5.49%/year, 4.45%/year and 2.16%/year respectively in those prescribed no antithrombotic therapy, aspirin, and warfarin. Similar trends were observed in patients with acute coronary syndromes. Diabetes mellitus, smoking history and renal failure requiring dialysis were predictors for primary endpoint in all antithrombotic therapies.
Conclusion
In patients with non-valvular atrial fibrillation, there is a modest association with coronary artery disease. Patients prescribed warfarin had the lowest risk of new onset coronary artery disease.
doi:10.1371/journal.pone.0131479
PMCID: PMC4476741  PMID: 26098876
17.  Potent Paracrine Effects of human induced Pluripotent Stem Cell-derived Mesenchymal Stem Cells Attenuate Doxorubicin-induced Cardiomyopathy 
Scientific Reports  2015;5:11235.
Transplantation of bone marrow mesenchymal stem cells (BM-MSCs) can protect cardiomyocytes against anthracycline-induced cardiomyopathy (AIC) through paracrine effects. Nonetheless the paracrine effects of human induced pluripotent stem cell-derived MSCs (iPSC-MSCs) on AIC are poorly understood. In vitro studies reveal that doxorubicin (Dox)-induced reactive oxidative stress (ROS) generation and cell apoptosis in neonatal rat cardiomyocytes (NRCMs) are significantly reduced when treated with conditioned medium harvested from BM-MSCs (BM-MSCs-CdM) or iPSC-MSCs (iPSC-MSCs-CdM). Compared with BM-MSCs-CdM, NRCMs treated with iPSC-MSCs-CdM exhibit significantly less ROS and cell apoptosis in a dose-dependent manner. Transplantation of BM-MSCs-CdM or iPSC-MSCs-CdM into mice with AIC remarkably attenuated left ventricular (LV) dysfunction and dilatation. Compared with BM-MSCs-CdM, iPSC-MSCs-CdM treatment showed better alleviation of heart failure, less cardiomyocyte apoptosis and fibrosis. Analysis of common and distinct cytokines revealed that macrophage migration inhibitory factor (MIF) and growth differentiation factor-15 (GDF-15) were uniquely overpresented in iPSC-MSC-CdM. Immunodepletion of MIF and GDF-15 in iPSC-MSCs-CdM dramatically decreased cardioprotection. Injection of GDF-15/MIF cytokines could partially reverse Dox-induced heart dysfunction. We suggest that the potent paracrine effects of iPSC-MSCs provide novel “cell-free” therapeutic cardioprotection against AIC, and that MIF and GDF-15 in iPSC-MSCs-CdM are critical for these enhanced cardioprotective effects.
doi:10.1038/srep11235
PMCID: PMC4460911  PMID: 26057572
18.  Multipotent (adult) and pluripotent stem cells for heart regeneration: what are the pros and cons? 
Heart failure after myocardial infarction is the leading cause of mortality and morbidity worldwide. Existing medical and interventional therapies can only reduce the loss of cardiomyocytes during myocardial infarction but are unable to replenish the permanent loss of cardiomyocytes after the insult, which contributes to progressive pathological left ventricular remodeling and progressive heart failure. As a result, cell-based therapies using multipotent (adult) stem cells and pluripotent stem cells (embryonic stem cells or induced pluripotent stem cells) have been explored as potential therapeutic approaches to restore cardiac function in heart failure. Nevertheless, the optimal cell type with the best therapeutic efficacy and safety for heart regeneration is still unknown. In this review, the potential pros and cons of different types of multipotent (adult) stem cells and pluripotent stem cells that have been investigated in preclinical and clinical studies are reviewed, and the future perspective of stem cell-based therapy for heart regeneration is discussed.
doi:10.1186/scrt381
PMCID: PMC4056686  PMID: 24476362
19.  Hypoadiponectinemia As an Independent Predictor for the Progression of Carotid Atherosclerosis: A 5-Year Prospective Study 
Abstract
Background: Hypoadiponectinemia predicts the development of diabetes and hypertension, both being potent atherosclerotic risk factors. Whether adiponectin predicts the progression of early atherosclerosis remains unclear. In this 5-year prospective study, we examined the relationship between serum adiponectin and carotid intima media thickness (CIMT), a marker of subclinical atherosclerosis.
Methods: A total of 265 subjects from the population-based Hong Kong Cardiovascular Risk Factor Prevalence Study, with no known cardiovascular disease, underwent CIMT measurement at baseline and at 5 years.
Results: In all, 129 men and 136 women, aged 54.6±12.3 years, were studied. Median CIMT at baseline was 0.63 mm (interquartile range 0.52–0.73 mm) and increased to 0.67 mm (0.56–0.78 mm) after 5 years (P<0.001). CIMT increment correlated with baseline adiponectin, age, and smoking (all P<0.05) and baseline CIMT (P<0.001), but not with sex, fasting glucose, lipid profiles, hypertension, or diabetes. In multiple linear regression analysis, baseline serum adiponectin level was an independent predictor of CIMT increment β (standardized beta)=−0.17, P=0.015], after adjusting for age, smoking, baseline CIMT, hypertension, body mass index, fasting glucose, low-density lipoprotein cholesterol, and triglycerides.
Conclusion: Hypoadiponectinemia predicted CIMT progression, independent of known predictive factors such as age, smoking, hyperlipidemia, and hypertension.
doi:10.1089/met.2014.0024
PMCID: PMC4241893  PMID: 25211296
20.  Clinical Potentials of Cardiomyocytes Derived from Patient-Specific Induced Pluripotent Stem Cells 
Journal of Clinical Medicine  2014;3(4):1105-1123.
The lack of appropriate human cardiomyocyte-based experimental platform has largely hindered the study of cardiac diseases and the development of therapeutic strategies. To date, somatic cells isolated from human subjects can be reprogramed into induced pluripotent stem cells (iPSCs) and subsequently differentiated into functional cardiomyocytes. This powerful reprogramming technology provides a novel in vitro human cell-based platform for the study of human hereditary cardiac disorders. The clinical potential of using iPSCs derived from patients with inherited cardiac disorders for therapeutic studies have been increasingly highlighted. In this review, the standard procedures for generating patient-specific iPSCs and the latest commonly used cardiac differentiation protocols will be outlined. Furthermore, the progress and limitations of current applications of iPSCs and iPSCs-derived cardiomyocytes in cell replacement therapy, disease modeling, drug-testing and toxicology studies will be discussed in detail.
doi:10.3390/jcm3041105
PMCID: PMC4470173  PMID: 26237594
patient-specific-iPSCs-derived cardiomyocytes; regenerative medicine; modeling of inherited cardiac disorder; drug testing; toxicology studies
21.  Continuation of Dabigatran Therapy in “Real-World” Practice in Hong Kong 
PLoS ONE  2014;9(8):e101245.
Background
Dabigatran, an oral direct thrombin inhibitor, possesses several advantages over warfarin that can in principle simplify the management of stroke prevention in atrial fibrillation (AF). Nonetheless it remains unclear whether these advantages can translate to clinical practice and encourage long-term therapy. The objective was to describe long-term dabigatran therapy for stroke prevention in AF and to identify risk factors for discontinuation of therapy.
Methods and Results
We studied 467 consecutive Chinese patients (72±11 years, male: 53.8%) with a mean CHA2DS2-VASc score of 3.6 prescribed dabigatran for stroke prevention in AF from March 2010 to September 2013. Over a mean follow-up of 16 months, 101 patients (21.6%) permanently discontinued dabigatran. The mean time-to-discontinuation was 8 months. The most common reason for discontinuation was dyspepsia (30.7%), followed by other adverse events (17.8%) such as minor bleeding (8.9%), major gastrointestinal bleeding (7.9%), and intracranial hemorrhage (1%). Other reasons included dosing frequency (5.9%), fear of side effects (4.0%), lack of laboratory monitoring (1.0%), and cost (1.0%). Multivariable analysis revealed that low baseline estimated glomerular filtration rate (p = 0.02), absence of hypertension (p = 0.01), and prior use of a proton-pump inhibitor (p = 0.02) and H2-receptor blocker (p = 0.01) were independent predictors of drug discontinuation. In addition, there were altogether 9 ischemic strokes (1.5%/years), 3 intracranial hemorrhages (0.5%/year), and 24 major gastrointestinal bleedings (4.1%/year).
Conclusion
Dabigatran discontinuation is very common amongst Chinese AF patients. This reveals a management gap in the prevention of stroke in AF.
doi:10.1371/journal.pone.0101245
PMCID: PMC4118845  PMID: 25084117
22.  Androgen Deprivation Therapy and Cardiovascular Risk in Chinese Patients with Nonmetastatic Carcinoma of Prostate 
Journal of Oncology  2014;2014:529468.
Background. Androgen deprivation therapy (ADT) in nonmetastatic prostate cancer is unclear. Recent data suggests possible increase in the cardiovascular risks receiving ADT. The aim of the study was to investigate the cardiovascular outcomes in a cohort of Chinese nonmetastatic prostate cancer patients with no previously documented cardiovascular disease. Methods and Results. 745 patients with no previously documented cardiovascular disease and/or diabetes mellitus diagnosed to have nonmetastatic prostate cancer were recruited. Of these, 517 patients received ADT and the remaining 228 did not. After a mean follow-up of 5.3 years, 60 patients developed primary composite endpoint including (1) coronary artery disease, (2) congestive heart failure, and (3) ischemic stroke. Higher proportion of patients on ADT (51 patients, 9.9%) developed composite endpoint compared with those not on ADT (9 patients, 3.9%) with hazard ratio (HR) of 2.06 (95% confidence interval (CI): 1.03–3.24, P = 0.04). Furthermore, Cox regression analysis revealed that only the use of ADT (HR: 2.1, 95% CI: 1.03–4.25, P = 0.04) and hypertension (HR: 2.0, 95% CI: 1.21–3.33, P < 0.01) were independent predictors for primary composite endpoint. Conclusion. ADT in Chinese patients with nonmetastatic prostate cancer with no previously documented cardiovascular disease was associated with subsequent development of cardiovascular events.
doi:10.1155/2014/529468
PMCID: PMC3997904  PMID: 24803931
23.  Predictive value of high-sensitivity troponin-I for future adverse cardiovascular outcome in stable patients with type 2 diabetes mellitus 
Introduction
High-sensitivity cardiac troponin I(hs-TnI) and T levels(hs-TnT) are sensitive biomarkers of cardiomyocyte turnover or necrosis. Prior studies of the predictive role of hs-TnT in type 2 diabetes mellitus(T2DM) patients have yielded conflicting results. This study aimed to determine whether hs-TnI, which is detectable in a higher proportion of normal subjects than hsTnT, is associated with a major adverse cardiovascular event(MACE) in T2DM patients.
Methods and results
We compared hs-TnI level in stored serum samples from 276 consecutive patients (mean age 65 ± 10 years; 57% male) with T2DM with that of 115 age-and sex-matched controls. All T2DM patients were prospectively followed up for at least 4 years for incidence of MACE including heart failure(HF), myocardial infarction(MI) and cardiovascular mortality. At baseline, 274(99%) patients with T2DM had detectable hs-TnI, and 57(21%) had elevated hs-TnI (male: 8.5 ng/L, female: 7.6 ng/L, above the 99th percentile in healthy controls). A total of 43 MACE occurred: HF(n = 18), MI(n = 11) and cardiovascular mortality(n = 14). Kaplan-Meier analysis showed that an elevated hs-TnI was associated with MACE, HF, MI and cardiovascular mortality. Although multivariate analysis revealed that an elevated hs-TnI independently predicted MACE, it had limited sensitivity(62.7%) and positive predictive value(38.5%). Contrary to this, a normal hs-TnI level had an excellent negative predictive value(92.2%) for future MACE in patients with T2DM.
Conclusion
The present study demonstrates that elevated hs-TnI in patients with T2DM is associated with increased MACE, HF, MI and cardiovascular mortality. Importantly, a normal hs-TnI level has an excellent negative predictive value for future adverse cardiovascular events during long-term follow-up.
doi:10.1186/1475-2840-13-63
PMCID: PMC4006634  PMID: 24661773
Type 2 diabetes mellitus; High-sensitivity troponin I outcome
24.  Mesenchymal Stem Cells Modulate Albumin-Induced Renal Tubular Inflammation and Fibrosis 
PLoS ONE  2014;9(3):e90883.
Bone marrow-derived mesenchymal stem cells (BM-MSCs) have recently shown promise as a therapeutic tool in various types of chronic kidney disease (CKD) models. However, the mechanism of action is incompletely understood. As renal prognosis in CKD is largely determined by the degree of renal tubular injury that correlates with residual proteinuria, we hypothesized that BM-MSCs may exert modulatory effects on renal tubular inflammation and epithelial-to-mesenchymal transition (EMT) under a protein-overloaded milieu. Using a co-culture model of human proximal tubular epithelial cells (PTECs) and BM-MSCs, we showed that concomitant stimulation of BM-MSCs by albumin excess was a prerequisite for them to attenuate albumin-induced IL-6, IL-8, TNF-α, CCL-2, CCL-5 overexpression in PTECs, which was partly mediated via deactivation of tubular NF-κB signaling. In addition, albumin induced tubular EMT, as shown by E-cadherin loss and α-SMA, FN and collagen IV overexpression, was also prevented by BM-MSC co-culture. Albumin-overloaded BM-MSCs per se retained their tri-lineage differentiation capacity and overexpressed hepatocyte growth factor (HGF) and TNFα-stimulating gene (TSG)-6 via P38 and NF-κB signaling. Albumin-induced tubular CCL-2, CCL-5 and TNF-α overexpression were suppressed by recombinant HGF treatment, while the upregulation of α-SMA, FN and collagen IV was attenuated by recombinant TSG-6. Neutralizing HGF and TSG-6 abolished the anti-inflammatory and anti-EMT effects of BM-MSC co-culture in albumin-induced PTECs, respectively. In vivo, albumin-overloaded mice treated with mouse BM-MSCs had markedly reduced BUN, tubular CCL-2 and CCL-5 expression, α-SMA and collagen IV accumulation independent of changes in proteinuria. These data suggest anti-inflammatory and anti-fibrotic roles of BM-MSCs on renal tubular cells under a protein overloaded condition, probably mediated via the paracrine action of HGF and TSG-6.
doi:10.1371/journal.pone.0090883
PMCID: PMC3960109  PMID: 24646687
25.  Worsened arterial stiffness in high-risk cardiovascular patients with high habitual carbohydrate intake: a cross-sectional vascular function study 
Background
Previous studies suggested that high dietary carbohydrate intake is associated with increased cardiovascular risk through raised triglyceride and decreased high-density lipoprotein-cholesterol levels. However, the relation between carbohydrate intake and arterial stiffness has not been established. The purpose of this study was to examine this relation among high-risk cardiovascular patients.
Methods
We studied the relation between dietary macronutrient intake and arterial stiffness in 364 patients with documented cardiovascular diseases or risk equivalent (coronary artery diseases 62%, ischemic stroke 13%, diabetes mellitus 55%) and in 93 age-and-sex matched control subjects. Dietary macronutrient intake was assessed using a validated food-frequency questionnaire (FFQ) for Chinese. Heart-ankle pulse wave velocity (PWV) was measured non-invasively with a Vascular Profiling System (VP2000, Colin Corp. USA). A dietary pattern with ≥60% total energy intake derived from carbohydrates was defined as a high-carbohydrate diet according to the Dietary Reference Intakes (DRI) for Chinese.
Results
Subjects who consumed a high-carbohydrate diet had significantly higher mean PWV than those who did not consume a high-carbohydrate diet (P = 0.039). After adjustment for potential confounders, high-carbohydrate diet was associated with significantly increased PWV [B = 73.50 (10.81 to 136.19), P = 0.022]. However, there was no significant association between high-carbohydrate diet and PWV in controls (P = 0.634).
Conclusions
High-carbohydrate diet is associated with increased arterial stiffness in patients with established cardiovascular disease or risk equivalent.
doi:10.1186/1471-2261-14-24
PMCID: PMC3948104  PMID: 24559092
Macronutrient; Carbohydrate intake; Arterial stiffness; Pulse wave velocity; Secondary prevention

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