Single-cell analysis reveals landscape of endometrial cancer response to estrogen and identification of early diagnostic markers.

BACKGROUND: The development of endometrial cancer (EC) is closely related to the abnormal activation of the estrogen signaling pathway. Effective diagnostic markers are important for the early detection and treatment of EC. METHOD: We downloaded single-cell RNA sequencing (scRNA-seq) and spatial transcriptome (ST) data of EC from public databases. Enrichment scores were calculated for EC cell subpopulations using the "AddModuleScore" function and the AUCell package, respectively. Six predictive models were constructed, including logistic regression (LR), Gaussian naive Bayes (GaussianNB), k-nearest neighbor (KNN), support vector machine (SVM), extreme gradient boosting (XGB), and neural network (NK). Subsequently, receiver-operating characteristics with areas under the curves (AUCs) were used to assess the robustness of the predictive model. RESULT: We classified EC cell coaggregation into six cell clusters, of which the epithelial, fibroblast and endothelial cell clusters had higher estrogen signaling pathway activity. We founded the epithelial cell subtype Epi cluster1, the fibroblast cell subtype Fib cluster3, and the endothelial cell subtype Endo cluster3 all showed early activation levels of estrogen response. Based on EC cell subtypes, estrogen-responsive early genes, and genes encoding Stage I and para-cancer differentially expressed proteins in EC patients, a total of 24 early diagnostic markers were identified. The AUCs values of all six classifiers were higher than 0.95, which indicates that the early diagnostic markers we screened have superior robustness across different classification algorithms. CONCLUSION: Our study elucidates the potential biological mechanism of EC response to estrogen at single-cell resolution, which provides a new direction for early diagnosis of EC.

Prohibitin 1 inhibits cell proliferation and induces apoptosis via the p53-mediated mitochondrial pathway in vitro.

BACKGROUND: Prohibitin 1 (PHB1) has been identified as an antiproliferative protein that is highly conserved and ubiquitously expressed, and it participates in a variety of essential cellular functions, including apoptosis, cell cycle regulation, proliferation, and survival. Emerging evidence indicates that PHB1 may play an important role in the progression of hepatocellular carcinoma (HCC). However, the role of PHB1 in HCC is controversial. AIM: To investigate the effects of PHB1 on the proliferation and apoptosis of human HCC cells and the relevant mechanisms in vitro. METHODS: HCC patients and healthy individuals were enrolled in this study according to the inclusion and exclusion criteria; then, PHB1 levels in the sera and liver tissues of these participates were determined using ELISA, RT-PCR, and immunohistochemistry. Human HepG2 and SMMC-7721 cells were transfected with the pEGFP-PHB1 plasmid and PHB1-specific shRNA (shRNA-PHB1) for 24-72 h. Cell proliferation was analysed with an MTT assay. Cell cycle progression and apoptosis were analysed using flow cytometry (FACS). The mRNA and protein expression levels of the cell cycle-related molecules p21, Cyclin A2, Cyclin E1, and CDK2 and the cell apoptosis-related molecules cytochrome C (Cyt C), p53, Bcl-2, Bax, caspase 3, and caspase 9 were measured by real-time PCR and Western blot, respectively. RESULTS: Decreased levels of PHB1 were found in the sera and liver tissues of HCC patients compared to those of healthy individuals, and decreased PHB1 was positively correlated with low differentiation, TNM stage III-IV, and alpha-fetoprotein ≥ 400 µg/L. Overexpression of PHB1 significantly inhibited human HCC cell proliferation in a time-dependent manner. FACS revealed that the overexpression of PHB1 arrested HCC cells in the G0/G1 phase of the cell cycle and induced apoptosis. The proportion of cells in the G0/G1 phase was significantly increased and the proportion of cells in the S phase was decreased in HepG2 cells that were transfected with pEGFP-PHB1 compared with untreated control and empty vector-transfected cells. The percentage of apoptotic HepG2 cells that were transfected with pEGFP-PHB1 was 15.41% ± 1.06%, which was significantly greater than that of apoptotic control cells (3.65% ± 0.85%, P < 0.01) and empty vector-transfected cells (4.21% ± 0.52%, P < 0.01). Similar results were obtained with SMMC-7721 cells. Furthermore, the mRNA and protein expression levels of p53, p21, Bax, caspase 3, and caspase 9 were increased while the mRNA and protein expression levels of Cyclin A2, Cyclin E1, CDK2, and Bcl-2 were decreased when PHB1 was overexpressed in human HCC cells. However, when PHB1 was upregulated in human HCC cells, Cyt C expression levels were increased in the cytosol and decreased in the mitochondria, which indicated that Cyt C had been released into the cytosol. Conversely, these effects were reversed when PHB1 was knocked down. CONCLUSION: PHB1 inhibits human HCC cell viability by arresting the cell cycle and inducing cell apoptosis via activation of the p53-mediated mitochondrial pathway.

Associations of psychological wellbeing with COVID-19 hospitalization and mortality in adults aged 50 years or older from 25 European countries and Israel.

Background: Lower psychological wellbeing is associated with poor outcomes in a variety of diseases and healthy populations. However, no study has investigated whether psychological wellbeing is associated with the outcomes of COVID-19. This study aimed to determine whether individuals with lower psychological wellbeing are more at risk for poor outcomes of COVID-19. Methods: Data were from the Survey of Health, Aging, and Retirement in Europe (SHARE) in 2017 and SHARE's two COVID-19 surveys in June-September 2020 and June-August 2021. Psychological wellbeing was measured using the CASP-12 scale in 2017. The associations of the CASP-12 score with COVID-19 hospitalization and mortality were assessed using logistic models adjusted for age, sex, body mass index, smoking, physical activity, household income, education level, and chronic conditions. Sensitivity analyses were performed by imputing missing data or excluding cases whose diagnosis of COVID-19 was solely based on symptoms. A confirmatory analysis was conducted using data from the English Longitudinal Study of Aging (ELSA). Data analysis took place in October 2022. Results: In total, 3,886 individuals of 50 years of age or older with COVID-19 were included from 25 European countries and Israel, with 580 hospitalized (14.9%) and 100 deaths (2.6%). Compared with individuals in tertile 3 (highest) of the CASP-12 score, the adjusted odds ratios (ORs) of COVID-19 hospitalization were 1.81 (95% CI, 1.41-2.31) for those in tertile 1 (lowest) and 1.37 (95% CI, 1.07-1.75) for those in tertile 2. As for COVID-19 mortality, the adjusted ORs were 2.05 (95% CI, 1.12-3.77) for tertile 1 and 1.78 (95% CI, 0.98-3.23) for tertile 2, compared with tertile 3. The results were relatively robust to missing data or the exclusion of cases solely based on symptoms. This inverse association of the CASP-12 score with COVID-19 hospitalization risk was also observed in ELSA. Conclusion: This study shows that lower psychological wellbeing is independently associated with increased risks of COVID-19 hospitalization and mortality in European adults aged 50 years or older. Further study is needed to validate these associations in recent and future waves of the COVID-19 pandemic and other populations.

Construction and Analysis of Hepatocellular Carcinoma Prognostic Model Based on Random Forest.

Methods: Transcriptome data and clinical data of HCC were downloaded from the TCGA database. Screen important genes based on the random forest method, combined with differential expression genes (DEGs) to screen out important DEGs. The Kaplan‒Meier curve was used to evaluate its prognostic significance. Cox regression analysis was used to construct a survival prognosis prediction model, and the ROC curve was used to verify it. Finally, the mechanism of action was explored through GO and KEGG pathway enrichment and GeneMANIA coexpression analyses. Results: Seven important DEGs were identified, three were highly expressed and four were lowly expressed. Among them, GPRIN1, MYBL2, and GSTM5 were closely related to prognosis (P < 0.05). After the survival prognosis prediction model was established, the survival analysis showed that the survival time of the high-risk group was significantly shortened (P < 0.001), but the ROC analysis indicated that the model was not superior to staging. Twenty coexpressed genes were screened, and enrichment analysis indicated that glutathione metabolism was an important mechanism for these genes to regulate HCC progression. Conclusion: This study revealed the important DEGs affecting HCC progression and provided references for clinical assessment of patient prognosis and exploration of HCC progression mechanisms through the construction of predictive models and gene enrichment analysis.

Peoniflorin Preconditioning Protects Against Myocardial Ischemia/Reperfusion Injury Through Inhibiting Myocardial Apoptosis: RISK Pathway Involved.

Preconditioning with Peoniflorin, a component of traditional Chinese prescriptions, was proposed to be a potential strategy for cardioprotection against ischemia/reperfusion (I/R) injury. However, the cardioprotective effect of Peoniflorin preconditioning has not been thoroughly confirmed, and the underlying mechanism remains unclear. Here, we examined the cardioprotective effect and its mechanism of Peoniflorin preconditioning against myocardial I/R injury. Rats were subjected to 30 min of transient ischemia followed by 2 h of reperfusion with or without Peoniflorin (100 mg/kg) prior to reperfusion. Peoniflorin preconditioning significantly limited myocardial infarct size and reperfusion arrhythmias, as well as obviously attenuated the histomorphological and micromorphological damages induced by I/R injury. The reduced myocardial injury was also associated with the anti-apoptotic effect of Peoniflorin, as evidence by decreased TUNEL-positive cells, upregulation of BCL-2 expression, and downregulation of Bax and caspase-3 expression. In an effort to evaluate the mechanism responsible for the observed cardioprotective and anti-apoptotic effect, Western blot of phosphorylated protein was performed after 20 min of reperfusion. Results showed that Peoniflorin preconditioning activated both the Akt and ERK1/2 arm of the reperfusion injury salvage kinase (RISK) pathway. To further confirm this mechanism, the PI3K signaling inhibitor LY294002 and ERK1/2 signaling inhibitor PD98059 were administered in vivo. The cardioprotective and anti-apoptotic effects of Peoniflorin preconditioning were diminished but not abolished by pretreatment with LY294002 or PD98059. Taken together, these results indicate that Peoniflorin preconditioning protects the myocardial against I/R injury and inhibits myocardial apoptosis via the activation of the RISK pathway, highlighting the potential therapeutic effects of Peoniflorin on reducing myocardial I/R injury.

Effectiveness of enhanced recovery after surgery in the perioperative management of patients with bone surgery in China.

BACKGROUND: Enhanced recovery after surgery (ERAS) was introduced in China in 2007. Over time, the scope of ERAS has expanded from abdominal surgery to orthopedics, urology and other fields. Continuous development and research has contributed to progress of ERAS in China. In 2019, to promote the application of ERAS in bone tumor surgery, we formed the "Consensus of Experts on Perioperative Management of Accelerated Rehabilitation in Major Surgery of Bone Tumors in China". AIM: To evaluate the effect of enhanced recovery after bone tumor surgery in perioperative management in China. METHODS: One hundred and seven patients who underwent bone tumor surgery at the Second Affiliated Hospital of Xi'an Jiaotong University between May 2019 and April 2021 were randomized into a study group (53 cases) and a control group (54 cases). The study group adopted the ERAS protocol and the control group adopted conventional care. Main outcome measures included postoperative length of stay (LOS), postoperative complications, mortality, and 30-d readmission rates. Secondary outcomes included postoperative visual analog scale (VAS) score of pain, number of blood transfusions, drainage volume in 24 h after operation, patient satisfaction 30 d after discharge, VAS score at 30 d after discharge, and daily standing walking time. RESULTS: There were no significant differences in the baseline data, clinical features and surgical site between the two groups. The LOS in the study group with the ERAS protocol was 7.72 ± 3.34 d compared with 10.28 ± 4.27 d in the control group who followed conventional care. The incidence of postoperative nausea and vomiting (PONV) in the study group was 19% and 37% in the control group. The VAS scores of pain on postoperative day 1 (POD1) and POD3 in the study group were 4.79 ± 2.34 and 2.79 ± 1.53 compared with 5.28 ± 3.27 and 3.98 ± 2.27 in the control group. The drainage volume in 24 h after the operation was 124.36 ± 23.43 mL in the study group and 167.43 ± 30.87 mL in the control group. The number of blood transfusions in the study group was also lower. The patient satisfaction rate was higher in the study group than in the control group. CONCLUSION: The ERAS protocol in the perioperative period of bone tumor surgery can decrease LOS, PONV, and postoperative pain, blood transfusion and 24-h drainage, improve patient satisfaction and accelerate recovery.

Irisin attenuates angiotensin II-induced cardiac fibrosis via Nrf2 mediated inhibition of ROS/ TGFß1/Smad2/3 signaling axis.

Angiotensin II-related cardiac fibrosis is one of the key pathological changes of the hypertrophied left ventricle in various heart disease. Irisin was recently reported to confer cardio-protective and anti-oxidative effects, while whether it can reverse the renin-angiotensin-aldosterone system(RAAS) activation related(angiotensin II-induced) cardiac fibrosis is unknown. In this study, we found that angiotensin II-induced cardiac dysfunction and fibrotic responses were dampened by irisin treatment in mice. Mechanistically, angiotensin II induced robust ROS generation, which in turn triggered activation of pro-fibrotic TGFß1-Smad2/3 signaling and subsequent collagen synthesis and fibroblast-myofibroblast transformation in cardiac fibroblasts. In contrast, Irisin treatment suppressed angiotensin II-induced ROS generation, TGFß1 activation, collagen synthesis and fibroblast-myofibroblast transformation, the effects of which was accompanied by Nrf2 activation and also abolished by a Nrf2 targeted siRNA. Taken together, we here identified irisin as a promising anti-fibrotic therapeutic for angiotensin II-related cardiac fibrosis.

Association of serum adropin with the presence of atrial fibrillation and atrial remodeling.

OBJECTIVE: Adropin, a newly identified regulatory protein encoded by Enho gene, suppressed tumor necrosis factor α-induced THP1 monocyte adhesion to human umbilical vein endothelial cells. In addition, inflammation is demonstrated to be involved in the mechanism of atrial fibrillation (AF). Atrial remodeling is correlated with the persistence and progression of AF. Adropin is hypothesized to correlated with AF and atrial remodeling. This study aims to determine the correlation of serum adropin and the presence of AF and remodeling. METHODS: This study consisted of 344 AF patients and 210 healthy controls. AF patients were then divided into three subgroups of paroxysmal AF, persistent AF, and permanent AF. Serum adropin concentrations were examined using enzyme-linked immunosorbent assay method. Left atrial diameter (LAD) was measured to evaluate atrial remodeling. RESULTS: Decreased serum adropin concentrations were found in AF patients compared with healthy controls. Logistic regression analysis confirmed that serum adropin was inversely associated with the presence of AF (OR 0.218, 95% CI 0.15-0.316; P < 0.001). Permanent AF patients had significantly reduced serum adropin concentrations compared with persistent and paroxysmal AF patients. There were decreased serum adropin concentrations in persistent AF group than those in paroxysmal AF group. Simple linear regression analyses showed that serum adropin in AF patients were negatively correlated with BMI, SBP, and LAD. Multiple stepwise regression analysis showed that LAD remained to be inversely associated with serum adropin (ß = 0.2, P = 0.010). CONCLUSION: Serum adropin concentrations are inversely correlated with the presence of AF and atrial remodeling.

Cilostazol suppresses angiotensin II-induced apoptosis in endothelial cells.

Patients with essential hypertension undergo endothelial dysfunction, particularly in the conduit arteries. Cilostazol, a type III phosphodiesterase inhibitor, serves a role in the inhibition of platelet aggregation and it is widely used in the treatment of peripheral vascular diseases. Previous studies have suggested that cilostazol suppresses endothelial dysfunction; however, it remains unknown whether cilostazol protects the endothelial function in essential hypertension. The aim of the present study was to investigate whether, and how, cilostazol suppresses angiotensin II (angII)­induced endothelial dysfunction. Human umbilical vein endothelial cells (HUVECs) and Sprague Dawley rats were exposed to angII and treated with cilostazol. Endothelial cell apoptosis and function, nitric oxide and superoxide production, phosphorylation (p) of Akt, and caspase­3 protein expression levels were investigated. AngII exposure resulted in the apoptosis of endothelial cells in vitro and in vivo. In vitro, cilostazol significantly suppressed the angII­induced apoptosis of HUVECs; however, this effect was reduced in the presence of LY294002, a phosphoinositide 3 kinase (PI3K) inhibitor. Furthermore, cilostazol suppressed the angII­induced p­Akt downregulation and cleaved caspase­3 upregulation. These effects were also alleviated by LY294002. In vivo, cilostazol suppressed the angII­induced endothelial cell apoptosis and dysfunction. Cilostazol was also demonstrated to partially reduced the angII­induced increase in superoxide production. The results of the present study suggested that cilostazol suppresses endothelial apoptosis and dysfunction by modulating the PI3K/Akt pathway.

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

BACKGROUND: Environmental 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. RESULTS: Here, 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/ß-catenin signaling by the selective antagonist Dickkopf-1 significantly reduced the activity of Wnt/ß-catenin signaling and decreased the cardiomyocyte differentiation of miPSCs cultured on the 3D PCL nanofibrous scaffold, while the early activation of Wnt/ß-catenin signaling by CHIR99021 further increased the cardiomyocyte differentiation of miPSCs. CONCLUSION: These 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/ß-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/ß-catenin signaling, which will be helpful in guiding future stem cell- and scaffold-based myocardium bioengineering.

Low-Level Electrical Stimulation of Aortic Root Ventricular Ganglionated Plexi Attenuates Autonomic Nervous System-Mediated Atrial Fibrillation.

OBJECTIVES: This study investigated the effect of electrical stimulation of aortic root ventricular ganglionated plexi (GP) on atrial fibrillation (AF) inducibility. BACKGROUND: The ventricular GP are interconnected with atrial GP to govern heart function, although the effect of ventricular GP modification on control of AF remains unknown. METHODS: Effective refractory periods (ERPs) of test pulmonary veins (PVs) were measured at baseline and during high-level (HL-ES) and low-level (LL-ES) electrical stimulation of the aortic root GP. The arrhythmogenic threshold of acetylcholine and isoproterenol was determined at baseline and during HL-ES and LL-ES. Moreover, AF was induced at PVs by programmed electrical stimulation after HL-ES or LL-ES. Immunohistochemistry staining was performed to examine the autonomic activity from aortic root GP to the PVs. RESULTS: Compared with the baseline group, HL-ES of aortic root GP significantly shortened atrial ERP (95 ± 13 ms vs. 122 ± 9 ms) and PV ERP (104 ± 11 ms vs. 131 ± 12 ms); decreased the threshold concentration of AF by both acetylcholine (1.3 ± 0.2 µmol/l vs. 3.2 ± 0.3 µmol/l) and isoproterenol (0.3 ± 0.1 µmol/l vs. 1.3 ± 0.2 µmol/l); and increased the AF-inducing rate from PVs (90% vs. 30%). In contrast, LL-ES of the GP prevented the shortening of ERP and PV ERP to 125 ± 10 ms and 133 ± 11 ms, respectively; increased threshold levels of acetylcholine and isoproterenol to 5.7 ± 0.4 µmol/l and 3.2 ± 0.3 µmol/l; and decreased the AF-inducing rate to 5%. We also found that the biotinylated dextran amine-containing varicose fibers projected directly from the aortic root GP to the left PVs. CONCLUSIONS: These findings suggest that autonomic innervations of left PVs partly originated from aortic root ventricular GP. Moreover, LL-ES of aortic root ventricular GP suppressed AF inducibility and arose from PVs mediated by the autonomic nervous system.

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.

Collagen/ß(1) integrin interaction is required for embryoid body formation during cardiogenesis from murine induced pluripotent stem cells.

BACKGROUND: The 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. RESULTS: In the present study, collagen type I and ß(1) 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/ß(1) integrin interaction by ß(1) 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. CONCLUSIONS: These findings indicate that collagen/ß(1) 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.

Activation of peripheral blood CD3(+) T-lymphocytes in patients with atrial fibrillation.

Atrial fibrillation (AF) is a common disease with a poorly understood pathophysiological mechanism. Increasing evidence indicates that AF may be associated with immunologic inflammation responses, but it remains unclear whether activation of peripheral blood CD3(+) T-lymphocytes plays a role in the pathogenesis of AF. The aim of this study was to evaluate this phenomenon. Fifty paroxysmal AF patients and 56 persistent AF patients who underwent successful electrical cardioversion were enrolled. The percentages of CD69 and human leukocyte antigen DR (HLA-DR) positive peripheral blood CD3(+) T-lymphocytes, which indicate T-lymphocyte activation, were examined by flow cytometric analysis in the patients and 51 healthy controls. The patient groups had higher levels of CD69 and HLA-DR than the healthy controls. During the 3-month follow-up, 37 patients had recurrence of AF (recurrence group) and 50 patients remained in sinus (sinus group). The CD69 and HLA-DR levels in the sinus group were all significantly down-regulated at follow-up compared with before cardioversion. However, there were no statistically significant differences between the CD69 and HLA-DR levels in the recurrence group at follow-up and before cardioversion. Our findings suggest that activation of peripheral blood CD3(+) T-lymphocytes was associated with AF, and might be a diagnostic or therapeutic marker.

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.

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.

Three-dimensional co-culture facilitates the differentiation of embryonic stem cells into mature cardiomyocytes.

The cardiomyocyte (CM) differentiation of embryonic stem cells (ESCs) is routinely cultured as two-dimensional (2D) monolayer, which doesn't mimic in vivo physiological environment and may lead to low differentiated level of ESCs. Here, we develop a novel strategy that enhances CM differentiation of ESCs in collagen matrix three-dimensional (3D) culture combined with indirect cardiac fibroblasts co-culture. ESCs were cultured in hanging drops to form embryoid bodies (EBs) and then applied on collagen matrix. The EBs were indirectly co-cultured with cardiac fibroblasts by the hanging cell culture inserts (PET 1 µm). The molecular expressions and ultrastructural characteristics of ESC-derived CMs (ESCMs) were analyzed by real time RT-PCR, immunocytochemistry, and Transmission Electron Microscopy (TEM). We found that the percentage of beating EBs with cardiac fibroblasts co-culture was significantly higher than that without co-culture after differentiation period of 8 days. Type I collagen used as 3D substrates enhanced the late-stage CM differentiation of ESCs and had effect on ultrastructural mature of ESCMs in late-stage development. The combined effects of 3D and co-culture that mimic in vivo physiological environment further improved the efficiency of CM differentiation from ESCs, resulting in fiber-like structures of cardiac cells with organized sarcomeric structure in ESCMs. This novel 3D co-culture system emphasizes the fact that the ESC differentiation is actively responding to cues from their environment and those cues can drive phenotypic control, which provides a useful in vitro model to investigate CM differentiation of stem cells.

Postprocedural serum sLOX-1 levels are associated with coronary in-stent restenosis in patients with stable coronary artery disease.

OBJECTIVES: We tested whether serum soluble lectin-like oxidized low-density lipoprotein receptor-1 (sLOX-1) levels are able to predict in-stent restenosis (ISR) after successful primary percutaneous coronary intervention (PCI). METHODS: Preprocedural and postprocedural serum sLOX-1 levels were measured in 210 consecutive patients with stable coronary artery disease who underwent successful primary PCI for de novo lesions. The patients were grouped as ISR and non-ISR based on angiographic follow-up results. RESULTS: PCI significantly increased serum sLOX-1 levels both in patients with [0.85 (range: 0.63-0.98) vs. 0.39 (range: 0.27-0.54) ng/ml, P < 0.01] or without ISR [0.45 (range: 0.36-0.84) vs. 0.32 (range: 0.28-0.62) ng/ml, P < 0.01]. Postprocedural serum sLOX-1 levels were higher in patients with ISR than those without ISR [0.85 (range: 0.63-0.98) vs. 0.45 (range: 0.36-0.84) ng/ml, P < 0.01]. High postprocedural serum sLOX-1 levels served as independent predictors of ISR (odds ratio: 3.040, 95% confidence interval: 1.359-6.802, P < 0.01). Furthermore, postprocedural serum sLOX-1 levels were correlated with late lumen loss of the stented lesions (ρ = 0.36, P < 0.01). CONCLUSION: Postprocedural serum sLOX-1 levels are significantly associated with the risk of ISR and the severity of lumen loss in patients with stable coronary artery disease undergoing primary PCI. These results suggested that postprocedural serum sLOX-1 levels might be useful for the detection and risk assessment of ISR after PCI.