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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.  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
7.  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
8.  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
9.  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
10.  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
11.  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
12.  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
13.  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
14.  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
15.  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
16.  Stroke Patients with a Past History of Cancer Are at Increased Risk of Recurrent Stroke and Cardiovascular Mortality 
PLoS ONE  2014;9(2):e88283.
Background and Purpose
Cancer patients are at increased risk of cardiovascular and cerebrovascular events. It is unclear whether cancer confers any additional risk for recurrent stroke or cardiovascular mortality after stroke.
Methods
This was a single center, observational study of 1,105 consecutive Chinese ischemic stroke patients recruited from a large stroke rehabilitation unit based in Hong Kong. We sought to determine whether patients with cancer are at higher risk of recurrent stroke and cardiovascular mortality.
Results
Amongst 1,105 patients, 58 patients (5.2%) had cancer, of whom 74% were in remission. After a mean follow-up of 76±18 months, 241 patients developed a recurrent stroke: 22 in patients with cancer (38%, annual incidence 13.94%/year), substantially more than those without cancer (21%, 4.65%/year) (p<0.01). In a Cox regression model, cancer, age and atrial fibrillation were the 3 independent predictors of recurrent stroke with a hazard ratio (HR) of 2.42 (95% confidence interval (CI): 1.54–3.80), 1.01 (1.00–1.03) and 1.35 (1.01–1.82) respectively. Likewise, patients with cancer had a higher cardiovascular mortality compared with those without cancer (4.30%/year vs. 2.35%/year, p = 0.08). In Cox regression analysis, cancer (HR: 2.08, 95% CI: 1.08–4.02), age (HR: 1.04, 95% CI 1.02–1.06), heart failure (HR: 3.06, 95% CI 1.72–5.47) and significant carotid atherosclerosis (HR: 1.55, 95% CI 1.02–2.36) were independent predictors for cardiovascular mortality.
Conclusions
Stroke patients with a past history of cancer are at increased risk of recurrent stroke and cardiovascular mortality.
doi:10.1371/journal.pone.0088283
PMCID: PMC3921146  PMID: 24523883
17.  An Upregulation in the Expression of Vanilloid Transient Potential Channels 2 Enhances Hypotonicity-Induced Cytosolic Ca2+ Rise in Human Induced Pluripotent Stem Cell Model of Hutchinson Gillford Progeria 
PLoS ONE  2014;9(1):e87273.
Hutchinson-Gillford Progeria Syndrome (HGPS) is a fatal genetic disorder characterized by premature aging in multiple organs including the skin, musculoskeletal and cardiovascular systems. It is believed that an increased mechanosensitivity of HGPS cells is a causative factor for vascular cell death and vascular diseases in HGPS patients. However, the exact mechanism is unknown. Transient receptor potential (TRP) channels are cationic channels that can act as cellular sensors for mechanical stimuli. The aim of this present study was to examine the expression and functional role of TRP channels in human induced pluripotent stem cell-derived endothelial cells (iPSC-ECs) from the patients with HGPS. The mRNA and protein expression of TRP channels in HGPS and control (IMR90) iPSC-ECs were examined by semi-quantitative RT-PCRs and immunoblots, respectively. Hypotonicity-induced cytosolic Ca2+ ([Ca2+]i) rise in iPSC-ECs was measured by confocal microscopy. RT-PCRs and immunoblots showed higher expressional levels of TRPV2 in iPSC-ECs from HGPS patients than those from normal individuals. In functional studies, hypotonicity induced a transient [Ca2+]i rise in iPSC-ECs from normal individuals but a sustained [Ca2+]i elevation in iPSC-ECs from HGPS patients. A nonselective TRPV inhibitor, ruthenium red (RuR, 20 µM), and a specific TRPV2 channel inhibitor, tranilast (100 µM), abolished the sustained phase of hypotonicity-induced [Ca2+]i rise in iPSC-ECs from HGPS patients, and also markedly attenuated the transient phase of the [Ca2+]i rise in these cells. Importantly, a short 10 min hypotonicity treatment caused a substantial increase in caspase 8 activity in iPSC-ECs from HGPS patients but not in cells from normal individuals. Tranilast could also inhibit the hypotonicity-induced increase in caspase 8 activity. Taken together, our data suggest that an up-regulation in TRPV2 expression causes a sustained [Ca2+]i elevation in HGPS-iPSC-ECs under hypotonicity, consequently resulting in apoptotic cell death. This mechanism may contribute to the pathogenesis of vascular diseases in HGPS patients.
doi:10.1371/journal.pone.0087273
PMCID: PMC3903625  PMID: 24475260
18.  A-FABP and Oxidative Stress Underlie the Impairment of Endothelium-Dependent Relaxations to Serotonin and the Intima-Medial Thickening in the Porcine Coronary Artery with Regenerated Endothelium 
ACS Chemical Neuroscience  2012;4(1):122-129.
Experiments were designed to determine the cause of the selective dysfunction of Gi proteins, characterized by a reduced endothelium-dependent relaxation to serotonin (5-hydroxytryptamine), in coronary arteries lined with regenerated endothelial cells. Part of the endothelium of the left anterior descending coronary artery of female pigs was removed in vivo to induce regeneration. The animals were treated chronically with vehicle (control), apocynin (antioxidant), or BMS309403 (A-FABP inhibitor) for 28 days before functional examination and histological analysis of segments of coronary arteries with native or regenerated endothelium of the same hearts. Isometric tension was recorded in organ chambers and cumulative concentration-relaxation curves obtained in response to endothelium-dependent [serotonin (Gi protein mediated activation of eNOS) and bradykinin (Gq protein mediated activation of eNOS)] and independent [detaNONOate (cGMP-mediated), isoproterenol (cAMP-mediated)] vasodilators. The two inhibitors tested did not acutely affect relaxations of preparations with either native or regenerated endothelium. In the chronically treated groups, however, both apocynin and BMS309403 abolished the reduction in relaxation to serotonin in segments covered with regenerated endothelium and prevented the intima-medial thickening caused by endothelial regeneration, without affecting responses to bradykinin or endothelium-independent agonists (detaNONOate and isoproterenol). Thus, inhibition of either oxidative stress or A-FABP likely prevents both the selective dysfunction of Gi protein mediated relaxation to serotonin and the neointimal thickening resulting from endothelial regeneration.
doi:10.1021/cn3000873
PMCID: PMC3547481  PMID: 23336051
Apocynin; BMS309403; bradykinin; endothelial cells; Gi proteins
19.  Long-term clinical outcomes of drug-eluting stents vs. bare-metal stents in Chinese geriatric patients 
Background & objective
Little is known about the relative efficacies of percutaneous coronary intervention (PCI) with drug-eluting stents (DES) and bare-metal stents (BMS) in elderly patients. The objective of this study was to evaluate the clinical outcome for geriatric patients who received either DES or BMS.
Methods
From January 2002 to October 2005, 199 consecutive Chinese geriatric patients (≥ 75 years old) underwent PCI with coronary DES or BMS implantation at our institution. We analyzed the major clinical end points that included all-cause mortality, cardiovascular death, myocardial infarction, target lesion revascularization (TLR), stent thrombosis, and bleeding complications.
Results
The three-year cumulative rates of all-cause mortality, cardiovascular death, and myocardial infarction were significantly lower in the DES group (6.3%, 3.6%, 5.4%) compared with the BMS group (16.2%, 11.5%, 14.9%; P < 0.05). No significant differences were found in the three-year cumulative rate for target lesion revascularization (6.3% vs. 4.6%, P = 0.61) or stent thrombosis (3.6% vs. 2.3%, P = 0.70). Likewise, there were no statistically significant differences in the cumulative rate for intracranial hemorrhage, or major and minor hemorrhage at three years.
Conclusions
DES-based PCI was associated with a significant reduction in the three-year cumulative rate of all-cause mortality, cardiovascular death, and myocardial infarction compared with BMS, without increased risk of TLR, stent thrombosis, or bleeding complications at three years in this group of Chinese geriatric patients.
doi:10.3969/j.issn.1671-5411.2013.04.003
PMCID: PMC3888914  PMID: 24454325
Percutaneous coronary intervention; Geriatric patients; Drug-eluting stents; Bare-metal stents
20.  Association of subclinical myocardial injury with arterial stiffness in patients with type 2 diabetes mellitus 
Objective
Type 2 diabetes mellitus (T2DM) is associated with subclinical myocardial injury although the underlying mechanism is uncertain. We postulated that arterial stiffness, endothelial dysfunction and subclinical atherosclerosis may contribute to subclinical myocardial injury in patients with T2DM.
Methods
Serum high-sensitivity troponin I (hs-TNI) an indicator of myocardial injury, was measured in 100 patients with T2DM without clinical evidence of macrovascular disease and 150 age and gender-matched controls. Elevated hs-TnI was defined as follow (derived from the 99th percentile from controls): Male >11.1 ng/L; female >7.6 ng/L. Measures that may contribute to myocardial damage in patients with T2DM, including brachial-ankle pulse wave velocity (ba-PWV), brachial flow mediated dilatation (FMD) and carotid intima media thickness (IMT), were also assessed.
Results
The serum level of hs-TNI (5.7±9.2 μg/L vs. 3.2±1.9 μg/L, P< 0.01) and the prevalence of elevated hs-TNI (12% vs. 4%, P = 0.02) were significantly higher in patients with T2DM than controls. Patients with T2DM also had significantly worse ba-PWV (17.98±3.91ms-1 vs. 15.70±2.96 ms-1), brachial FMD (2.6±3.5% vs. 5.5±4.2%, P< 0.01) and carotid IMT (0.96±0.20 mm vs. 0.86±0.14 mm, P< 0.01). In patients with T2DM, hs-TNI was positively correlated with systolic blood pressure (r = 0.31, P<0.01), serum creatinine (r = 0.26, P = 0.01) and ba-PWV (r = 0.34, P< 0.01). Importantly, multiple regression revealed that only ba-PWV was independently associated with hs-TNI (β = 0.25, P = 0.04).
Conclusion
The results demonstrated an independent association between ba-PWV and hs-TNI in patients with T2DM with no clinical evidence of macrovascular disease. These findings suggest that increased arterial stiffness is closely related to subclinical myocardial injury in patients with T2DM.
doi:10.1186/1475-2840-12-94
PMCID: PMC3706358  PMID: 23799879
Type 2 diabetes mellitus; Myocardial injury; Arterial stiffiness; High-sensitivity troponin I
21.  Attenuation of Hind-Limb Ischemia in Mice with Endothelial-Like Cells Derived from Different Sources of Human Stem Cells 
PLoS ONE  2013;8(3):e57876.
Functional endothelial-like cells (EC) have been successfully derived from different cell sources and potentially used for treatment of cardiovascular diseases; however, their relative therapeutic efficacy remains unclear. We differentiated functional EC from human bone marrow mononuclear cells (BM-EC), human embryonic stem cells (hESC-EC) and human induced pluripotent stem cells (hiPSC-EC), and compared their in-vitro tube formation, migration and cytokine expression profiles, and in-vivo capacity to attenuate hind-limb ischemia in mice. Successful differentiation of BM-EC was only achieved in 1/6 patient with severe coronary artery disease. Nevertheless, BM-EC, hESC-EC and hiPSC-EC exhibited typical cobblestone morphology, had the ability of uptaking DiI-labeled acetylated low-density-lipoprotein, and binding of Ulex europaeus lectin. In-vitro functional assay demonstrated that hiPSC-EC and hESC-EC had similar capacity for tube formation and migration as human umbilical cord endothelial cells (HUVEC) and BM-EC (P>0.05). While increased expression of major angiogenic factors including epidermal growth factor, hepatocyte growth factor, vascular endothelial growth factor, placental growth factor and stromal derived factor-1 were observed in all EC cultures during hypoxia compared with normoxia (P<0.05), the magnitudes of cytokine up-regulation upon hypoxic were more dramatic in hiPSC-EC and hESC-EC (P<0.05). Compared with medium, transplanting BM-EC (n = 6), HUVEC (n = 6), hESC-EC (n = 8) or hiPSC-EC (n = 8) significantly attenuated severe hind-limb ischemia in mice via enhancement of neovascularization. In conclusion, functional EC can be generated from hECS and hiPSC with similar therapeutic efficacy for attenuation of severe hind-limb ischemia. Differentiation of functional BM-EC was more difficult to achieve in patients with cardiovascular diseases, and hESC-EC or iPSC-EC are readily available as “off-the-shelf” format for the treatment of tissue ischemia.
doi:10.1371/journal.pone.0057876
PMCID: PMC3589485  PMID: 23472116
22.  Elevated Circulating Adipocyte‐Fatty Acid Binding Protein Levels Predict Incident Cardiovascular Events in a Community‐Based Cohort: A 12‐Year Prospective Study 
Background
Obesity is closely associated with various cardiovascular diseases (CVDs). Adipose tissue inflammation and perturbation of adipokine secretion may contribute to the pathogenesis of CVD. This study aimed to evaluate whether the 2 most abundant adipokines, adipocyte‐fatty acid binding protein (A‐FABP) and adiponectin, are independent risk factors predisposing to CVD.
Method and Results
We investigated prospectively the 12‐year development of CVD in relation to the baseline levels of A‐FABP and adiponectin in a population‐based community cohort comprising 1847 Chinese subjects recruited from the Hong Kong Cardiovascular Risk Factors Prevalence Study 2 (CRISPS 2) cohort without previous CVD. Baseline serum levels of A‐FABP, adiponectin, and C‐reactive protein (CRP), an established biomarker predictive of CVD, were measured. In all, 182 (9.9%) of the 1847 Chinese subjects developed CVD during a median follow‐up of 9.4 years. The CVD group had more traditional risk factors, higher baseline levels of A‐FABP and CRP (both P<0.001), but similar adiponectin levels (P=0.881) compared with the non‐CVD group. In Cox regression analysis including both biomarkers, the adjusted HR for A‐FABP and CRP for subjects above the optimal cutoff values were 1.57 (95% CI, 1.14 to 2.16; P=0.006) and 1.60 (95% CI, 1.12 to 2.27; P=0.01), respectively, after adjustment for traditional risk factors. The category‐free net reclassification index, but not the c‐statistic, showed improvement in predictive performance by the addition of A‐FABP to the traditional risk factor model (P=0.017).
Conclusions
Circulating A‐FABP level predicts the development of CVD after adjustment for traditional risk factors in a community‐based cohort. Its clinical use for CVD prediction warrants further validation.
doi:10.1161/JAHA.112.004176
PMCID: PMC3603238  PMID: 23525430
adipocyte‐fatty acid binding protein; adipokines; adiponectin; cardiovascular diseases; inflammation
23.  Myocardial dysfunction in patients with type 2 diabetes mellitus: role of endothelial progenitor cells and oxidative stress 
Background
Endothelial progenitor cells (EPCs) are responsible for angiogenesis and maintenance of microvascular integrity, the number of EPCs is correlated with oxidative stress. Their relation to myocardial dysfunction in patients with type 2 diabetes mellitus (T2DM) is nonetheless unknown.
Methods
Eighty-seven patients with T2DM and no history of coronary artery disease were recruited. Transthoracic echocardiography and detailed evaluation of left ventricular (LV) systolic function by 2-dimensional (2D) speckle tracking derived strain analysis in 3 orthogonal directions was performed. Four subpopulations of EPCs, including CD34+, CD133+, CD34+/kinase insert domain-containing receptor (KDR) + and CD133+/KDR + EPCs, were measured by flow cytometry. Oxidative stress was assessed by superoxide dismutase (SOD).
Results
The mean age of the patients was 62 ± 9 years and 39.6% were male. Those with an impaired longitudinal strain had a lower number of CD34+ EPCs (2.82 ± 1.87% vs. 3.74 ± 2.12%, P < 0.05) than those with preserved longitudinal strain. When compared with those with preserved circumferential strain, patients with an impaired circumferential strain had a lower number of CD34+ EPCs (2.63 ± 1.80% vs. 3.87 ± 2.10%, P < 0.01) and SOD level (0.13 ± 0.06U/ml vs. 0.20 ± 0.08U/ml, P < 0.01). Patients with an impaired radial strain nonetheless had a lower number of CD34+ EPCs (2.62 ± 2.08% vs. 3.69 ± 1.99%, P < 0.05). Multivariate analysis demonstrated that only impaired global circumferential strain remained significantly associated with CD34 + EPCs and SOD.
Conclusions
LV global circumferential strain was independently associated with number of CD34+ EPCs and SOD. These findings suggest that myocardial dysfunction in patients with T2DM is related to depletion of EPCs and increased oxidative stress.
doi:10.1186/1475-2840-11-147
PMCID: PMC3537556  PMID: 23217199
Type 2 diabetes mellitus; Myocardial injury; Endothelial progenitor cells
24.  Altered profile of circulating endothelial progenitor cells in obstructive sleep apnea 
Background
Obstructive sleep apnea (OSA) is independently associated with endothelial dysfunction, which may be perpetuated by alteration in endothelial repair capacity. Our study evaluates changes in endothelial progenitor cell (EPC) profile in relation to OSA and the role of advanced glycation end-products (AGE) in this relationship.
Methods
Consecutive Chinese adults undergoing sleep studies, who had no medical illnesses or regular medications, were enrolled. Subjects with morbid obesity or grossly elevated lipoprotein levels were excluded from analysis. Circulating EPC was measured with flow cytometry analysis.
Results
Seventy-two subjects, 64 % with OSA defined by apnea–hypopnea index (AHI) ≥ 5, were analyzed. CD34+ cell counts were positively correlated with oxygen desaturation index (ODI) (r = 0.250, p = 0.041) and duration of oxygen desaturation <90 % (T90) (r = 0.261, p = 0.033) and negatively with minimal oxygen saturation (r = −0.247, p = 0.044) after adjusting for age, glucose, body weight, and smoking status. AGE was positively correlated with indices of OSA severity (AHI, r = 0.249, p = 0.042; ODI, r = 0.244, p = 0.047; T90, r = 0.243, p = 0.047; minimal oxygen saturation, r = −0.251, p = 0.041) and negatively with CD133+ cells (r = −0.281, p = 0.021). On stepwise multiple linear regression analysis, minimal oxygen saturation (p = 0.013) and CD133+ cell counts (p = 0.029) were found to be significant determinants of AGE level (R2 = 0.147).
Conclusions
Nocturnal hypoxemia in OSA subjects was associated with increase in endothelial cells (CD34+) which may promote vascular repair. Accumulation of AGE in OSA may lead to diminution in early EPC (CD133+) and endothelial repair capacity over time, thus contributing to vascular pathogenesis.
doi:10.1007/s11325-012-0781-4
PMCID: PMC3742956  PMID: 23179139
Advanced glycation end-products; Endothelial progenitor cells; Intermittent hypoxia
25.  Prognostic implications of surrogate markers of atherosclerosis in low to intermediate risk patients with Type 2 Diabetes 
Background
Type 2 diabetes mellitus (T2DM) patients are at increased risk of developing cardiovascular events. Unfortunately traditional risk assessment scores, including the Framingham Risk Score (FRS), have only modest accuracy in cardiovascular risk prediction in these patients.
Methods
We sought to determine the prognostic values of different non-invasive markers of atherosclerosis, including brachial artery endothelial function, carotid artery atheroma burden, ankle-brachial index, arterial stiffness and computed tomography coronary artery calcium score (CACS) in 151 T2DM Chinese patients that were identified low-intermediate risk from the FRS recalibrated for Chinese (<20% risk in 10 years). Patients were prospectively followed-up and presence of atherosclerotic events documented for a mean duration of 61 ± 16 months.
Results
A total of 17 atherosclerotic events in 16 patients (11%) occurred during the follow-up period. The mean FRS of the study population was 5.0 ± 4.6% and area under curve (AUC) from receiver operating characteristic curve analysis for prediction of atherosclerotic events was 0.59 ± 0.07 (P = 0.21). Among different vascular assessments, CACS > 40 had the best prognostic value (AUC 0.81 ± 0.06, P < 0.01) and offered significantly better accuracy in prediction compared with FRS (P = 0.038 for AUC comparisons). Combination of FRS with CACS or other surrogate vascular markers did not further improve the prognostic values over CACS alone. Multivariate Cox regression analysis identified CACS > 40 as an independent predictor of atherosclerotic events in T2DM patients (Hazards Ratio 27.11, 95% Confidence Interval 3.36-218.81, P = 0.002).
Conclusions
In T2DM patients identified as low-intermediate risk by the FRS, a raised CACS > 40 was an independent predictor for atherosclerotic events.
doi:10.1186/1475-2840-11-101
PMCID: PMC3444371  PMID: 22900680
Vascular markers of atherosclerosis; Type 2 diabetes mellitus

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