Xiong-Tao Liu

Serum omentin-1 levels are inversely associated with the presence and severity of coronary artery disease in patients with metabolic syndrome

Context: Omentin-1, an adipokine secreted from visceral adipose tissue, has been reported to be associated with coronary artery disease (CAD) and metabolic disorders. Objective: To clarify the relationship between serum omentin-1 levels and the presence and severity of CAD in patients with metabolic syndrome (MetS). Methods: We measured serum omentin-1 levels in 175 consecutive patients with MetS and in 46 controls. Results: Serum omentin-1 levels are inversely associated with the presence and angiographic severity of CAD in MetS patients. Conclusions: Serum omentin-1 might be a potential biomarker to predict the development and progression of CAD in MetS patients.

Collagen/β1 integrin interaction is required for embryoid body formation during cardiogenesis from murine induced pluripotent stem cells

BackgroundThe interactions between stem cells and extracellular matrix (ECM) mediated by integrins play important roles in the processes that determine stem cell fate. However, the role of ECM/integrin interaction in the formation of embryoid bodies (EBs) during cardiogenesis from murine induced pluripotent stem cells (miPSCs) remains unclear.ResultsIn the present study, collagen type I and β1 integrin were expressed and upregulated synergistically during the formation of miPSC-derived EBs, with a peak expression at day 3 of differentiation. The blockage of collagen/β1 integrin interaction by β1 integrin blocking antibody resulted in the production of defective EBs that were characterized by decreased size and the absence of a shell-like layer composed of primitive endoderm cells. The quantification of spontaneous beating activity, cardiac-specific gene expression and cardiac troponin T (cTnT) immunostaining showed that the cardiac differentiation of these defective miPSC-derived EBs was lower than that of control EBs.ConclusionsThese findings indicate that collagen/β1 integrin interaction is required for the growth and cardiac differentiation of miPSC-derived EBs and will be helpful in future engineering of the matrix microenvironment within EBs to efficiently direct the cardiac fate of pluripotent stem cells to promote cardiovascular regeneration.

Three-dimensional poly-(ε-caprolactone) nanofibrous scaffolds directly promote the cardiomyocyte differentiation of murine-induced pluripotent stem cells through Wnt/β-catenin signaling

BackgroundEnvironmental factors are important for stem cell lineage specification, and increasing evidence indicates that the nanoscale geometry/topography of the extracellular matrix (ECM) directs stem cell fate. Recently, many three-dimensional (3D) biomimetic nanofibrous scaffolds resembling many characteristics of the native ECM have been used in stem cell-based myocardial tissue engineering. However, the biophysical role and underlying mechanism of 3D nanofibrous scaffolds in cardiomyocyte differentiation of induced pluripotent stem cells (iPSCs) remain unclear.ResultsHere, we fabricated a 3D poly-(ε-caprolactone) (PCL) nanofibrous scaffold using the electrospinning method and verified its nanotopography and porous structure by scanning electron microscopy. We seeded murine iPSCs (miPSCs) directly on the 3D PCL nanofibrous scaffold and initiated non-directed, spontaneous differentiation using the monolayer method. After the 3D PCL nanofibrous scaffold was gelatin coated, it was suitable for monolayer miPSC cultivation and cardiomyocyte differentiation. At day 15 of differentiation, miPSCs differentiated into functional cardiomyocytes on the 3D PCL nanofibrous scaffold as evidenced by positive immunostaining of cardiac-specific proteins including cardiac troponin T (cTnT) and myosin light chain 2a (MLC2a). In addition, flow cytometric analysis of cTnT-positive cells and cardiac-specific gene and protein expression of cTnT and sarcomeric alpha actinin (α-actinin) demonstrated that the cardiomyocyte differentiation of miPSCs was more efficient on the 3D PCL nanofibrous scaffold than on normal tissue culture plates (TCPs). Furthermore, early inhibition of Wnt/β-catenin signaling by the selective antagonist Dickkopf-1 significantly reduced the activity of Wnt/β-catenin signaling and decreased the cardiomyocyte differentiation of miPSCs cultured on the 3D PCL nanofibrous scaffold, while the early activation of Wnt/β-catenin signaling by CHIR99021 further increased the cardiomyocyte differentiation of miPSCs.ConclusionThese results indicated that the electrospun 3D PCL nanofibrous scaffolds directly promoted the cardiomyocyte differentiation of miPSCs, which was mediated by the activation of the Wnt/β-catenin signaling during the early period of differentiation. These findings highlighted the biophysical role of 3D nanofibrous scaffolds during the cardiomyocyte differentiation of miPSCs and revealed its underlying mechanism involving Wnt/β-catenin signaling, which will be helpful in guiding future stem cell- and scaffold-based myocardium bioengineering.

Increased Th17 cells in coronary artery disease are associated with neutrophilic inflammation

Abstract Objective. Atherosclerosis is a progressive disease characterized by a series of inflammatory responses in the large and medium arteries. Th17 cells, a distinct T cell lineage which has recently been identified, have a proinflammatory role and are implicated in many inflammatory conditions in humans and mice. The present study was designed to assess whether Th17 cells are associated with human coronary atherosclerosis. Design. Flow cytometry was used to examine Th17 cell frequencies in patients with coronary atherosclerosis and in healthy individuals. ELISA and real-time RT-PCR were performed to investigate circulating interleukin (IL)-17 (the signature cytokine of Th17 cells) and IL-8 (the cytokine induced by IL-17) protein and mRNA levels. Results. Significantly increased Th17 cell frequencies are observed in patients with coronary artery disease compared to healthy controls. The protein and mRNA levels of IL-17 and IL-8 are also significantly elevated in patients with atherosclerosis compared to healthy volunteers. Furthermore, mRNA levels of IL-17 and IL-8 are correlated with each other and with peripheral neutrophil counts. Conclusions. Our findings indicate that Th17 cells and their signature cytokine are involved in the process of atherogenesis. These data suggest that Th17 cells link T cell activity with neutrophilic inflammation in atherosclerosis.

High-Mobility Group Box 1 Induces Calcineurin-Mediated Cell Hypertrophy in Neonatal Rat Ventricular Myocytes

Cardiac hypertrophy is an independent predictor of cardiovascular morbidity and mortality. In recent years, evidences suggest that high-mobility group box 1 (HMGB1) protein, an inflammatory cytokine, participates in cardiac remodeling; however, the involvement of HMGB1 in the pathogenesis of cardiac hypertrophy remains unknown. The aim of this study was to investigate whether HMGB1 is sufficient to induce cardiomyocyte hypertrophy and to identify the possible mechanisms underlying the hypertrophic response. Cardiomyocytes isolated from 1-day-old Sprague-Dawley rats were treated with recombinant HMGB1, at concentrations ranging from 50 ng/mL to 200 ng/mL. After 24 hours, cardiomyocytes were processed for the evaluation of atrial natriuretic peptide (ANP) and calcineurin A expression. Western blot and real-time RT-PCR was used to detect protein and mRNA expression levels, respectively. The activity of calcineurin was also evaluated using a biochemical enzyme assay. HMGB1 induced cardiomyocyte hypertrophy, characterized by enhanced expression of ANP, and increased protein synthesis. Meanwhile, increased calcineurin activity and calcineurin A protein expression were observed in cardiomyocytes preconditioned with HMGB1. Furthermore, cyclosporin A pretreatment partially inhibited the HMGB1-induced cardiomyocyte hypertrophy. Our findings suggest that HMGB1 leads to cardiac hypertrophy, at least in part through activating calcineurin.

Involvement of reactive oxygen species and JNK in increased expression of MCP-1 and infiltration of inflammatory cells in pressure-overloaded rat hearts

Increasing evidence has shown that inflammation is involved in pressure overload-induced cardiac remodeling. Monocyte chemoattractant protein-1 (MCP-1) plays a pivotal role in the inflammatory process. However, the mechanisms underlying the upregulation of MCP-1 expression remain poorly understood. In the present study, we examined the hypothesis that an increased production of reactive oxygen species (ROS) mediates the upregulation of MCP-1. In a pressure-overloaded rat heart model with abdominal aortic coarctation (AC), superoxide dismutase-inhibitable cytochrome C reduction assay showed that ROS generation in the myocardium increased significantly at 1 week by 61% (n=8, P<0.01), peaked at 2 weeks and maintained these high levels for 4 weeks. The elevation of ROS was paralleled by the increased expression of MCP-1 and left ventricular remodeling (cardiac hypertrophy, perivascular and interstitial fibrosis). The oral administration of the antioxidant, N-acetylcysteine (NAC, 0.2 g/kg/day), for 2 or 4 weeks, significantly attenuated ROS production by 69 and 68%, respectively (n=8, P<0.01), as well as left ventricular remodeling. NAC treatment for 2 weeks also significantly reduced the MCP-1 mRNA and protein levels by 52 and 60%, respectively (n=4-8, both P<0.01), but had no effect on blood pressure. In the rats with AC at 2 weeks, when MCP-1 expression and inflammation changes were overt, immunoblotting with phospho-specific antibodies revealed that extracellular regulated kinase (ERK) and c-jun NH2-terminal kinase (JNK), but not p38 mitogen-activated protein kinase, were activated. NAC administration attenuated JNK activation, but had no effect on ERK. Our results suggest that increased ROS production may play an important role in the increased expression of MCP-1 in pressure overload-induced cardiac remodeling. JNK is likely involved in the signaling pathway.

Silencing GIRK4 expression in human atrial myocytes by adenovirus-delivered small hairpin RNA

GIRK4 has been shown to be a subunit of IKACh, and the use of GIRK4 in human atrial myocytes to treat arrhythmia remains an important research pursuit. Adenovirus-delivered small hairpin RNA (shRNA) has been used to mediate gene knockdown in mouse cardiocytes, yet there is no information on the successful application of this technique in human cardiocytes. In the current study, we used a siRNA validation system to select the most efficient sequence for silencing GIRK4. To this end, adenovirus-delivered shRNA, which expresses this sequence, was used to silence GIRK4 expression in human atrial myocytes. Finally, the feasibility, challenges, and results of silencing GIRK4 expression were evaluated by RT-PCR, western blotting, and the voltage-clamp technique. The levels of mRNA and protein were depressed significantly in cells infected by adenovirus-delivered shRNA against GIRK4, approximately 86.3% and 51.1% lower than those cells infected by adenovirus-delivered nonsense shRNA, respectively. At the same time, IKACh densities were decreased 53% by adenovirus-delivered shRNA against GIRK4. In summary, adenovirus-delivered shRNA against GIRK4 mediated efficient GIRK4 knockdown in human atrial myocytes and decreased IKACh densities. As such, these data indicated that adenovirus-delivered shRNA against GIRK4 is a potential tool for treating arrhythmia.

The Long-Term Differentiation of Embryonic Stem Cells into Cardiomyocytes: An Indirect Co-Culture Model

Background Embryonic Stem Cells (ESCs) can differentiate into cardiomyocytes (CMs) in vitro but the differentiation level from ESCs is low. Here we describe a simple co-culture model by commercially available Millicell™ hanging cell culture inserts to control the long-term differentiation of ESCs into CMs. Methodology/Principal Findings Mouse ESCs were cultured in hanging drops to form embryoid bodies (EBs) and treated with 0.1 mmol/L ascorbic acid to induce the differentiation of ESCs into CMs. In the indirect co-culture system, EBs were co-cultured with epidermal keratinocytes (EKs) or neonatal CMs (NCMs) by the hanging cell culture inserts (PET membranes with 1 µm pores). The molecular expressions and functional properties of ESC-derived CMs in prolonged culture course were evaluated. During time course of ESC differentiation, the percentages of EBs with contracting areas in NCMs co-culture were significantly higher than that without co-culture or in EKs co-culture. The functional maintenance of ESC-derived CMs were more prominent in NCMs co-culture model. Conclusions/Significance These results indicate that NCMs co-culture promote ESC differentiation and has a further effect on cell growth and differentiation. We assume that the improvement of the differentiating efficiency of ESCs into CMs in the co-culture system do not result from the effect of co-culture directly on cell differentiation, but rather by signaling effects that influence the cells in proliferation and long-term function maintenance.

Epicardial ablation of right pulmonary artery ganglionated plexi for the prevention of atrial fibrillation originating in the pulmonary veins

UNLABELLED PROBLEM PRESENTED: A novel study of catheter ablation of the right pulmonary artery ganglionated plexi (RPA GP) to reduce atrial fibrillation (AF) originating in the pulmonary veins (PVs) is presented. STUDIES UNDERTAKEN In 20 dogs, atrial effective refractory periods (AERPs), PVERP, and the dispersion of AERP (dAERP) were measured at baseline during RPA GP stimulation and after ablation. Programmed stimulation and burst stimulation protocols were performed at 4 distal PVs to measure the percentage of AF induced before and after ablation. RESULTS Stimulation of the RPA GP shortened AERP (116 +/- 16 vs 130 +/- 10 milliseconds, P < .01) and PVERP (122 +/- 14 vs 136 +/- 12 milliseconds, P < .01), and increased dAERP (31 +/- 6 vs 23 +/- 6 milliseconds, P < .01). However, the above indices revealed an adverse change after excision (AERP, 138 +/- 7 vs 130 +/- 10 milliseconds; PVERP, 146 +/- 18 vs 136 +/- 12 milliseconds; and dAERP, 19 +/- 5 vs 23 +/- 6 milliseconds; P < .05). Furthermore, the percentage of AF induced from PVs was significantly reduced with vagosympathetic stimulation (40% vs 90%, P < .01). CONCLUSIONS Ablation of the RPA GP changes the electrophysiologic properties of both the atria and the PVs and decreases AF inducibility arising from the PVs.

Simvastatin inhibits angiotensin II-induced cardiac cell hypertrophy: Role of Homer 1a

1 The scaffolding protein Homer 1a is constitutively expressed in the myocardium, although its function in cardiomyocytes remains poorly understood. The aim of the present study was to investigate Homer 1a expression in hypertrophic cardiac cells and its role in angiotensin (Ang) II‐induced cardiac hypertrophy. 2 After serum starvation for 24 h, cells were treated with 1 μmol/L simvastatin, 100 nmol/L angiotensin (Ang) II or their combination added to Dulbeccos modified Eagles medium containing 0.5% serum. For combination treatment with AngII plus simvastatin, cells were exposed to simvastatin 12 h before the addition of AngII to the medium and cells were then incubated in the presence of both drugs for a further 24 h. Western blotting was used to determine Homer 1a protein expression. Hypertrophy was evaluated by determining the protein content per cell. 3 Homer 1a protein levels were upregulated following AngII‐induced hypertrophy in H9C2 cells and neonatal rat cardiomyocytes, and these increases were augmented by simvastatin pretreatment. Concomitantly, simvastatin pretreatment inhibited extracellular signal‐regulated kinase (ERK) 1/2 phosphorylation and AngII‐induced hypertrophy. 4 The inhibitory effects of simvastatin against AngII‐induced hypertrophy were attenuated by Homer 1a silencing, suggesting that simvastatin suppresses cardiac hypertrophy in a Homer 1a‐dependent manner. Furthermore, AngII‐induced hypertrophy and ERK1/2 phosphorylation in neonatal rat cardiomyocytes were significantly inhibited following the overexpression of Homer 1a using an adenovirus. 5 These results suggest a possible role for Homer 1a in inhibiting cardiac hypertrophy perhaps in part through inhibition of ERK1/2 activation.