Safety and efficacy of autologous thymosin ß4 pre-treated endothelial progenitor cell transplantation in patients with acute ST segment elevation myocardial infarction: A pilot study.

BACKGROUND: Experimental studies and clinical trials suggest that endothelial progenitor cell (EPC) transplantation can repair "broken" heart. However, transplantation of autologous EPCs has numerous limitations, including the limited supply of expanded EPCs, the impaired function and activity of the transplanted cells and so on. Therefore, we investigated the feasibility, safety and initial clinical outcome of autologous thymosin ß4 (Tß4) pre-treated EPC transplantation in patients with acute ST segment elevation myocardial infarction (STEMI). METHODS: Ten patients with STEMI were included; they were randomized to 2 groups: EPC transplantation group (control group; n = 5) and Tß4-pre-treated EPC transplantation group (experimental group; n = 5). EPCs were pre-treated with Tß4 24 hours before transplantation in experimental group. Cardiac function was evaluated using echocardiography and emission computed tomography, as well as the 6-min walking test before and 6 months after the intervention. RESULTS: After 6 months of follow-up, the average 6-min walking distance was increased by 38.2 m (from 263 ± 42 m to 302 ± 34 m) in the control group and 75.7 m (from 264 ± 42 m to 340 ± 44 m) in the experimental group; the average difference of the 6-min walking distance was 37.5 m (95% confidence interval [CI], 28.7-56.3 m; P < 0.01). In addition, the cardiac function in the experimental group was more significantly improved than that of the control group. There were no severe complications related to the procedure in either group during the follow-up. DISCUSSION: Our pilot study suggested that Tß4-optimized EPC transplantation appeared to be feasible and safe, and might have beneficial effects on exercise capacity and left ventricular function in patients with STEMI.

Advanced Glycation End Product (AGE)-AGE Receptor (RAGE) System Upregulated Connexin43 Expression in Rat Cardiomyocytes via PKC and Erk MAPK Pathways.

The remodeling of cardiac gap junction contributes to the arrhythmias in a diabetic heart. We previously reported that high glucose reduced Cx43 protein level in neonatal rat cardiomyocytes. But, the effect and mechanisms of advanced glycation end product (AGE) on Cx43 expression still remain unclear. In this study, we measured the AGE receptor (RAGE) and Cx43 expression by immunohistochemisty in AGE-infused Sprague-Dawley (SD) rats. In vitro, the Cx43 and RAGE levels were detected in AGE-treated cardiomyocytes by Western blot and real-time RT-PCR. The function of cells coupling was measured by Scrap loading dye transfer assay. Our results showed that the AGE-infused rat hearts exhibited increased cardiac RAGE and Cx43, as well as Cx43 redistribution. In cultured cardiomyocytes, AGE elevated RAGE expression in a time- and dose-dependent manner. Cx43 protein and mRNA levels were upregulated by AGE (200 mg/L, 24 h), but the gap junction function was not enhanced. RAGE-targeted knock-down or the addition of PKC, and Erk inhibitors abolished the effect of AGE on Cx43. Therefore, AGE-RAGE system might elevate Cx43 expression in rat cardiomyocytes by activating PKC and Erk MAPK pathways, and it also enhanced Cx43 redistribution in vivo, which might contribute to the arrhythmias in diabetes.

Predicting the Prognosis of Patients in the Coronary Care Unit: A Novel Multi-Category Machine Learning Model Using XGBoost.

Background: Early prediction and classification of prognosis is essential for patients in the coronary care unit (CCU). We applied a machine learning (ML) model using the eXtreme Gradient Boosting (XGBoost) algorithm to prognosticate CCU patients and compared XGBoost with traditional classification models. Methods: CCU patients' data were extracted from the MIMIC-III v1.4 clinical database, and divided into four groups based on the time to death: <30 days, 30 days-1 year, 1-5 years, and ≥5 years. Four classification models, including XGBoost, naïve Bayes (NB), logistic regression (LR), and support vector machine (SVM) were constructed using the Python software. These four models were tested and compared for accuracy, F1 score, Matthews correlation coefficient (MCC), and area under the curve (AUC) of the receiver operating characteristic curves. Subsequently, Local Interpretable Model-Agnostic Explanations method was performed to improve XGBoost model interpretability. We also constructed sub-models of each model based on the different categories of death time and compared the differences by decision curve analysis. The optimal model was further analyzed using a clinical impact curve. At last, feature ablation curves of the XGBoost model were conducted to obtain the simplified model. Results: Overall, 5360 CCU patients were included. Compared to NB, LR, and SVM, the XGBoost model showed better accuracy (0.663, 0.605, 0.632, and 0.622), micro-AUCs (0.873, 0.811, 0.841, and 0.818), and MCC (0.337, 0.317, 0.250, and 0.182). In subgroup analysis, the XGBoost model had a better predictive performance in acute myocardial infarction subgroup. The decision curve and clinical impact curve analyses verified the clinical utility of the XGBoost model for different categories of patients. Finally, we obtained a simplified model with thirty features. Conclusions: For CCU physicians, the ML technique by XGBoost is a potential predictive tool in patients with different conditions, and it may contribute to improvements in prognosis.

Predictive value of the relative lymphocyte count in coronary heart disease.

The physiological stress suffered by patients with coronary heart disease (CHD) may result in a shift in leukocyte differential toward a decreased percentage of lymphocytes (L%). The purpose of this study was to determine the prognostic value of a low L% in CHD. One hundred forty patients evaluated in our department between 2007 and 2008 were retrospectively reviewed. Thirty-eight patients had primary percutaneous coronary intervention (PCI), and 102 patients had elective PCI. Various statistical analyses were used to examine the association between a low L% or other clinical characteristics and CHD. Univariate analysis showed that low L% was significantly related to ACS compared with stable CHD or control. White blood cell (WBC) count, C-reactive protein (CRP) and left ventricular systolic dysfunction (LVSD) were also correlated with CHD. Multivariate analysis and logistic regression analysis revealed that L%, CRP, WBC count and LVSD were all independently significant risk factors to have predictive value for CHD and 1 year major adverse cardiac events (MACE). A low L% could be used as an independent predictor for ACS on admission and is associated with MACE during clinical follow-up in CHD patients.

The Rho kinase inhibitor, fasudil, ameliorates diabetes-induced cardiac dysfunction by improving calcium clearance and actin remodeling.

Previous study showed inhibition of RhoA and Rho kinase (ROCK) activity with fasudil could alleviate diabetes-induced cardiac dysfunction partially due to improvement of myocardial fibrosis. However, the effect of fasudil on intracellular calcium cycling and actin remodeling, both of which are important to regulate excitation-contract coupling, is still not fully elucidated. In this study, a diabetic cardiomyopathy model was induced by a single intraperitoneal injection of streptozotocin (STZ) in male Sprague Dawley rats. Diabetic rats were treated with fasudil or placebo for 8 weeks. We found that long-term administration of fasudil, a specific Rho kinase inhibitor, significantly ameliorated diabetes-induced contractile dysfunction both at cellular and whole organ levels. Fasudil-treated rats displayed improved diastolic intracellular calcium ([Ca2+]i) removal and rescued expression of protein responsible for [Ca2+]i clearance. Furthermore, our study indicated that fasudil treatment normalized the phosphorylation of the PKCß2/Akt pathway in the diabetic heart, which might be the underlying mechanism accounting for the protective effect of fasudil on [Ca2+]i clearance. In addition, compared to the diabetes group, fasudil also normalized the G/F-actin ratio by preventing cofilin phosphorylation and promoted F-actin organization, suggesting a beneficial effect on actin remodeling. These findings indicate the protective effect of fasudil against diabetes-induced cardiac dysfunction via modulation of Ca2+ handling and actin remodeling. Overactivation of RhoA/ROCK plays a key role in the development of DCM. Inhibition of ROCK activity with fasudil improved [Ca2+]i removal in diabetic cardiomyocytes. Fasudil normalized the G/F-actin ratio and promoted F-actin organization. ROCK may be an excellent therapeutic target for the treatment of DCM. KEY MESSAGE: Overactivation of RhoA/ROCK plays a key role in the development of DCM. Inhibition of ROCK activity with fasudil improved [Ca2+]i removal in diabetic cardiomyocytes. Fasudil normalized the G/F-actin ratio and promoted F-actin organization. ROCK may be an excellent therapeutic target for the treatment of DCM.

Effect of radiofrequency catheter ablation on endothelial function and oxidative stress.

OBJECTIVE: Thromboembolic complications commonly occur in radiofrequency (RF) ablation procedures. Endothelial injury is believed to be induced during the process of RF ablation and to be involved in the mechanisms of thromboembolism. In this study, we gave further information about endothelial injury and also investiged the possible reasons for endothelial dysfunction during RF. METHODS AND RESULTS: Plasma levels of nitric oxide (NO), a biomarker of endothelial injury and some antioxidants and oxidants: beta-carotene (beta-CAR), lipoperoxide (LPO), superoxide dismutase (SOD), vitamin C (VC) and vitamin E (VE) were measured in 44 patients who underwent radiofrequency catheter ablation. Plasma NO concentration decreased significantly from 433.27 +/- 59.93 nmol/L to 407.13 +/- 52.32 nmol/L during RF ablation. Levels of beta-CAR, LPO, SOD,VC and VE were not significantly different. The decrease of plasma NO level was significantly correlated with ablation duration and cumulative ablation energy but had no significant correlation with overall procedure time, output ablation energy, ablation temperature and age of the patients. CONCLUSIONS: Radiofrequency catheter ablation induced endothelial injury but had no effect on oxidative stress. Radiofrequency energy was the main factor that caused endothelial injury during RF ablation.

NADPH oxidase activation played a critical role in the oxidative stress process in stable coronary artery disease.

OBJECTIVES: The study was designed to investigate the oxidative stress levels of endothelial progenitor cells (EPCs) in stable coronary artery disease (CAD) and to explore the underlying mechanisms of NADPH oxidase activation and subsequent EPCs dysfunction. METHODS: EPCs were isolated from patients with stable CAD (n=50) and matched healthy volunteers (n=50). NADPH oxidase activation was detected by measuring the expression of each subunit using western blotting and qPCR analyses and the membrane translocation of p47phox using immunofluorescence. The in vivo angiogenesis capacity was evaluated using immunofluorescence by transplanting EPCs into a rat hind limb ischemia model. The PKC inhibitor GÖ-6983 was used to determine the role of PKC in NADPH oxidase activation. RESULTS: Oxidative stress level was increased and the in vivo angiogenesis capacity was impaired in EPCs obtained from CAD subjects with the activation of NADPH oxidase. P47phox membrane translocation increased in CAD group vs controls. These effects were resolved by NADPH oxidase inhibition. Up-regulation of PKCα/ß2 was found in EPCs from CAD subjects, PKC inhibition GÖ-6983 could reduce the expression and activity of NADPH oxidation. CONCLUSIONS: NADPH oxidase activation via p47phox membrane translocation played a critical role in the initiation and progression of CAD, and the PKCα/ß2 signaling pathway might be involved.

[Sbudies on the effect of parthenolide on the proliferation of rat vascular smooth muscle cells and the mechanism].

OBJECTIVE: To study the effect of parthenolide on the proliferation of vascular smooth muscle cell(VSMC) and its mechanism. METHOD: Vascular smooth muscle cell was cultured, the protein levels of c-fos, c-myc, p15, p16, p18, p19 were measured by Western blot method, cell cycle were examined with flow cytometry, and the DNA synthesis was determined by [3H]-TdR incorporation. RESULT: Parthenolide inhibited protein levels of c-fos, c-myc in a time-dependent manner but didn't affect the protein levels of p15, p16, p18, p19. Flow cytometric DNA analysis revealed that parthenolide increased significantly G0/G1 phase of VSMC and decreased S phase of VSMC in a dose-dependent manner. Parthenolide inhibited [3H]-TdR incorporation in a dose dependent manner. CONCLUSION: Parthenolide may inhibit proliferation of VSMC by inhibiting the expressions of c-fos, c-myc, but not the expressions of p15, p16, p18, p19.

Protective effect of erythropoietin on myocardial infarction in rats by inhibition of caspase-12 expression.

In the present study, the myocardial protective effects of erythropoietin (EPO) by inhibition of the expression of caspase-12 were investigated in a myocardial infarction rat model. Thirty male SD rats were divided into three groups: sham-operation group, myocardial infarction group and EPO treatment group. The myocardial infarction model was created by ligating the left anterior descending coronary artery. The EPO treatment group was established by injecting rh-EPO (1,000 IU/kg) intraperitoneally every day after the operation, and the other two groups were injected with sodium chloride. Four weeks after induction of myocardial infarction, the left ventricular diastolic pressure (LVDP) was tested by Langendorff apparatus and the pathological changes were analyzed by H&E staining. Caspase-12 expression in the left ventricular myocardium was also measured by immunohistochemistry. Four weeks after induction of myocardial infarction, the improvement in heart function in the EPO treatment group was more distinct compared to that of the myocardial infarction group; LVDP was higher in the EPO treatment group compared to the myocardial infarction group, but lower compared to the control group. H&E staining showed that the myocardial cells in the normal control group were aligned in order with a clear structure and were stained equably, while the myocardial cells in the myocardial infarction model rats lined up in disorder with an augmented cell body appearing to have many granules and interstitial fibrosis. Myocardial fibrosis and disorder were improved in the EPO treatment group. The expression of caspase-12 in the myocardial infarction group was also increased compared to the EPO treatment group rats. The results suggest that EPO improves heart function in myocardial infarction rats by down-regulating the expression of caspase-12, which may protect the myocardium by abrogating endoplasmic reticulum stress-mediated cardiomyocyte apoptosis and improving heart function.

Enhanced collateral growth by double transplantation of gene-nucleofected fibroblasts in ischemic hindlimb of rats.

BACKGROUND: Induction of neovascularization by releasing therapeutic growth factors is a promising application of cell-based gene therapy to treat ischemia-related problems. In the present study, we have developed a new strategy based on nucleofection with alternative solution and cuvette to promote collateral growth and re-establishment of circulation in ischemic limbs using double transplantation of gene nucleofected primary cultures of fibroblasts, which were isolated from rat receiving such therapy. METHODS AND RESULTS: Rat dermal fibroblasts were nucleofected ex vivo to release bFGF or VEGF165 in a hindlimb ischemia model in vivo. After femoral artery ligation, gene-modified cells were injected intramuscularly. One week post injection, local confined plasmid expression and transient distributions of the plasmids in other organs were detected by quantitative PCR. Quantitative micro-CT analyses showed improvements of vascularization in the ischemic zone (No. of collateral vessels via micro CT: 6.8±2.3 vs. 10.1±2.6; p<0.05). Moreover, improved collateral proliferation (BrdU incorporation: 0.48±0.05 vs. 0.57±0.05; p<0.05) and increase in blood perfusion (microspheres ratio: gastrocnemius: 0.41±0.10 vs. 0.50±0.11; p<0.05; soleus ratio: soleus: 0.42±0.08 vs. 0.60±0.08; p<0.01) in the lower hindlimb were also observed. CONCLUSIONS: These results demonstrate the feasibility and effectiveness of double transplantation of gene nucleofected primary fibroblasts in producing growth factors and promoting the formation of collateral circulation in ischemic hindlimb, suggesting that isolation and preparation of gene nucleofected cells from individual accepting gene therapy may be an alternative strategy for treating limb ischemia related diseases.

Parthenolide inhibits proliferation of vascular smooth muscle cells through induction of G0/G1 phase cell cycle arrest.

OBJECTIVE: This study is to determine the effect of the natural product parthenolide, a sesquiterpene lactone isolated from extracts of the herb Tanacetum parthenium, on the proliferation of vascular smooth muscle cells (VSMCs). METHODS: Rat aortic VSMCs were isolated and cultured in vitro, and treated with different concentrations of parthenolide (10, 20 and 30 mumol/L). [(3)H]thymidine incorporation was used as an index of cell proliferation. Cell cycle progression and distribution were determined by flow cytometric analysis. Furthermore, the expression of several regulatory proteins relevant to VSMC proliferation including IkappaBalpha, cyclooxygenase-2 (Cox-2), p21, and p27 was examined to investigate the potential molecular mechanism. RESULTS: Treatment with parthenolide significantly decreased the [(3)H]thymidine incorporation into DNA by 30%~56% relative to control values in a dose-dependent manner (P<0.05). Addition of parthenolide also increased cell population at G(0)/G(1) phase by 19.2%~65.7% (P<0.05) and decreased cell population at S phase by 50.7%~84.8% (P<0.05), which is consistent with its stimulatory effects on p21 and p27. In addition, parthenolide also increased IkappaBalpha expression and reduced Cox-2 expression in a time-dependent manner. CONCLUSION: Our results show that parthenolide significantly inhibits the VSMC proliferation by inducing G(0)/G(1) cell cycle arrest. IkappaBalpha and Cox-2 are likely involved in such inhibitory effect of parthenolide on VSMC proliferation. These findings warrant further investigation on potential therapeutic implications of parthenolide on VSMC proliferation in vivo.