Characteristics of Wellens' Syndrome in the Current PCI Era: A Single-Center Retrospective Study.

Objectives: The goal of this retrospective study was to reveal the prevalence, angiographic characteristics, clinical presentation, and long-term outcomes of non-ST-segment elevation myocardial infarction (NSTEMI) patients with Wellens' syndrome. Background: Procedural results for percutaneous coronary intervention (PCI) in acute coronary syndrome (ACS) have improved in recent years. However, there is still a paucity of available clinical trial data for Wellens' syndrome even though it is a well-known high-risk ACS. Methods: Among a total of 3528 patients with ACS who underwent angioplasty from 2017 to 2019 at the Cardiovascular Center of Beijing Friendship Hospital, 476 NSTEMI patients with culprit left anterior descending (LAD) vessels were enrolled in this study. According to electrocardiographic criteria of Wellens' syndrome, the patients were divided into a Wellens group (n = 138) and a non-Wellens group (n = 338). The primary endpoint was cardiac death; the secondary endpoints were main adverse cardiovascular and cerebrovascular events (MACCEs), a composite of all-cause death, cardiac death, heart failure, target lesion revascularization, recurrent myocardial infarction, and stroke. All of the medical and follow-up data were obtained from our institutional database. Results: The incidence of Wellens' syndrome in all ACS patients was 5.7% (200 of 3528). Among the 200 patients with Wellens' syndrome, 138 had NSTEMI, for a proportion of 69%. There was a significant decrease in the percentage of preexisting coronary heart disease (CHD), prior myocardial infarction, and previous PCI (P < 0.05) in the Wellens group compared with the non-Wellens group. On coronary angiography, single-vessel lesions were more common in the Wellens group (11.6% vs. 5.3%, P=0.016), and almost all (97.1%) of these patients received drug-eluting stents. Notably, the Wellens group had a higher proportion of early PCI than the non-Wellens group (71% vs. 61.2%, P=0.044). At 24 months, there was no statistically significant difference in cardiac death (P=0.111) between the two groups, but the MACCEs were comparable (Wellens: 5.1% vs. non-Wellens: 13.3%, P=0.009). Age ≥65 years was the largest independent risk factor for adverse prognosis. Conclusions: With early recognition and aggressive intervention, Wellens' syndrome is no longer a risk factor for adverse prognosis in patients with NSTEMI in the current PCI era.

A practical and robust method for beam blocker-based cone beam CT scatter correction.

Objective. In the traditional beam-blocker based cone beam CT (CBCT) scatter correction, the scatter measured in the region shaded by lead strips was multiplied by a correction factor to directly represent the scatter in the unblocked region. The correction factor optimization is a tedious process and lacks an objective stop criterion. To skip the optimization process, an indirect scatter estimation method was developed and validated in phantom imaging.Approach.A beam-blocker made of lead strips was mounted between the x-ray source and object for scatter estimation. The primary signal between lead strips in the blocked region was first calculated by subtracting the measured scatter, and then used to calculate the scatter signal in the unblocked region corresponding to the same attenuation path. The calculated scatter signal was smoothed via local filtration and used to correct the measured projection in the unblocked region. Finally, the CBCT was reconstructed via Feldkamp-Davis-Kress algorithm. A Catphan and a head phantom were used to verify the performance of the proposed method in both full- and half-blocker scenarios, and with and without a bow-tie filter.Main Results. For scans without the bow-tie filter, the CT number error was reduced to 3.97±2.27 and 5.51±3.90 HU in the full- and half-blocker scenarios, respectively, for the Catphan, and to 4.01±2.18 and 7.97 ± 4.05 HU for the head phantom. When the bow-tie filter was applied, the CT number error was reduced to 2.29±1.42 and 6.72±0.77 HU in the full- and half-blocker scenarios, respectively, for the Catphan, and 2.35±1.25 and 4.96 ± 1.89 HU for the head phantom.Significance. The proposed method effectively avoids the influence of the inserted beam blocker itself on the scatter intensity estimation, and proves a more practical and robust way for the beam-blocker based scatter correction in CBCT scanning.

Tongxinluo attenuates atherosclerosis by inhibiting ROS/NLRP3/caspase-1-mediated endothelial cell pyroptosis.

ETHNOPHARMACOLOGICAL RELEVANCE: Tongxinluo (TXL) is one of the most common traditional Chinese medicines and plays a vital role in treating atherosclerosis (AS). Endothelial cell (EC) pyroptosis plays a crucial role in the development of AS. Previous research revealed the inhibitory function of TXL in EC apoptosis and autophagy. However, whether TXL can inhibit the pyroptosis of ECs has not been determined. AIM OF THE STUDY: To explore the influence of TXL on EC pyroptosis and determine its underlying mechanism of action in AS. MATERIALS AND METHODS: The TXL components were determined by ultra-performance liquid chromatography coupled with a photodiode array detector. We used ApoE-/- mice to establish a disease model of AS. After treatment with TXL, we recorded pathological changes in the mice and performed immunofluorescence staining of mice aortas. We also measured protein and gene levels to explore the influence of TXL on pyroptosis in vivo. The model was established by stimulating mouse aortic endothelial cells (MAECs) with oxidized low-density lipoprotein (ox-LDL) and analyzing the effect of TXL on pyroptosis by Western blotting (WB), real-time PCR (RT-PCR), and flow cytometry (FCM). We also investigated the impact of TXL on reactive oxygen species (ROS) by FCM and WB. RESULTS: Ten major components of TXL were detected. The vivo results showed that TXL inhibited the development of AS and decreased EC pyroptosis, the activation of caspase-1, and the release of inflammatory cytokines. The vitro experiments showed that TXL significantly reduced the extent of injury to MAECs by oxidized LDL (ox-LDL). TXL reversed the high expression of gasdermin D and other proteins induced by ox-LDL and had a significant synergistic effect with the caspase-1 inhibitor VX-765. We also confirmed that TXL decreased the accumulation of ROS and the expression levels of its essential regulatory proteins Cox2 and iNOS. When ROS accumulation was reduced, EC pyroptotic damage was reduced accordingly. CONCLUSION: Our results indicated that TXL inhibited EC pyroptosis in AS. Reducing the accumulation of ROS may be the essential mechanism of AS inhibition by TXL.

Development and validation of a scatter-corrected CBCT image-guided method for cervical cancer brachytherapy.

Background and purpose: Multiple patient transfers have a nonnegligible impact on the accuracy of dose delivery for cervical cancer brachytherapy. We consider using on-site cone-beam CT (CBCT) to resolve this problem. However, CBCT clinical applications are limited due to inadequate image quality. This paper implements a scatter correction method using planning CT (pCT) prior to obtaining high-quality CBCT images and evaluates the dose calculation accuracy of CBCT-guided brachytherapy for cervical cancer. Materials and methods: The CBCT of a self-developed female pelvis phantom and five patients was first corrected using empirical uniform scatter correction in the projection domain and further corrected in the image domain. In both phantom and patient studies, the CBCT image quality before and after scatter correction was evaluated with registered pCT (rCT). Model-based dose calculation was performed using the commercial package Acuros®BV. The dose distributions of rCT-based plans and corrected CBCT-based plans in the phantom and patients were compared using 3D local gamma analysis. A statistical analysis of the differences in dosimetric parameters of five patients was also performed. Results: In both phantom and patient studies, the HU error of selected ROIs was reduced to less than 15 HU. Using the dose distribution of the rCT-based plan as the baseline, the γ pass rate (2%, 2 mm) of the corrected CBCT-based plan in phantom and patients all exceeded 98% and 93%, respectively, with the threshold dose set to 3, 6, 9, and 12 Gy. The average percentage deviation (APD) of D90 of HRCTV and D2cc of OARs was less than 1% between rCT-based and corrected CBCT-based plans. Conclusion: Scatter correction using a pCT prior can effectively improve the CBCT image quality and CBCT-based cervical brachytherapy dose calculation accuracy, indicating promising prospects in both simplified brachytherapy processes and accurate brachytherapy dose delivery.

Neutrophil-to-lymphocyte ratio predicts coronary artery lesion severity and long-term cardiovascular mortality in patients with unstable angina pectoris.

OBJECTIVES: Neutrophil-to-lymphocyte ratio (NLR), one of the composite biomarker of systemic inflammatory status, was proved promising in predicting clinical outcomes of acute coronary syndrome (ACS). However, there were no evidences that NLR was directly relative to the clinical outcomes of unstable angina pectoris (UAP). Therefore, this study was aimed to detect whether NLR could predict the coronary artery lesion severity (indicated as SYNTAX score) and clinical outcomes (especially long-term cardiovascular mortality) in patients with. METHODS: In the single-centre retrospective study, 4110 patients with UAP were enrolled and divided into two groups according to their primary NLR values and followed up at a median time duration of 36 months. The differences of SYNTAX score and cardiovascular mortality between groups were analysed, and the predictive value of NLR was determined. RESULTS: NLR was positively and linearly correlated with SYNTAX score (r = 0.270). Diabetes (p = 0.049), lymphocyte (p = 0.004), NLR (p = 0.002) and SYNTAX score (p < 0.001) were independent predictors of long-term cardiovascular mortality in patients with UAP. Kaplan-Meier analysis revealed higher occurrence of cardiovascular mortality when NLR > 2.38 (p = 0.015). Receiver operating characteristic (ROC) analysis showed that NLR = 2.76 is an effective cut point for predicting cardiovascular mortality (69.2% sensitivity, 64.8% specificity). CONCLUSIONS: NLR value was positively related to the severity of coronary artery lesion and proved to be an independent predictor of cardiovascular mortality in patients with UAP. This study would contribute to therapy and prognosis optimisation of UAP.

The uric acid to albumin ratio: a novel predictor of long-term cardiac mortality in patients with unstable angina pectoris after percutaneous coronary intervention.

The prognosis of unstable angina pectoris (UAP) differs from non-ST-segment elevation myocardial infarction, and percutaneous coronary intervention (PCI) is considered to improve outcomes of UAP. This study aimed to assess the prognostic value of uric acid to albumin ratio (UAR) for long-term mortality in UAP patients after PCI. Our study retrospectively enrolled 2298 patients hospitalized because of UAP in a tertiary hospital. Divided by medium UAR, the patients were classified into two groups. Baseline demographics, clinical features and laboratory characteristics were obtained from medical records. Post-discharge follow-up was performed either in outdoor clinic or through phone call. The primary endpoint in this study was cardiac death, while all-cause death and rehospitalization were designated as the secondary endpoints. The median follow-up time was 672 days. Among all patients, 58 (2.5%) died, 28 of which died of cardiac deaths (1.2%), and 467 were re-hospitalized (20.3%). Cardiac mortality and all-cause mortality were found to be significantly higher in the high UAR group than in the low UAR group (p = 0.007, p < 0.001), and Kaplan-Meier analysis showed patients with higher UAR may suffer from worse outcomes (p = 0.020). UAR, PCI history, and age were identified as independent predictors of cardiac mortality by multivariate Cox regression. A UAR value of >8.35 was demonstrated as an ideal cut-off point to predict post-PCI cardiac mortality (p <0.001). Overall, it is indicated that baseline UAR was independently correlated with long-term cardiac mortality in patients with UAP treated by PCI.

Resveratrol regulates paracrine function of cardiac microvascular endothelial cells under hypoxia/reoxygenation condition.

The secreted factors from cardiac microvascular endothelial cells (CMECs) regulate the physiological activity of adjacent tissues and could be modulated by myocardial ischemia/reperfusion injury (MIRI). How this paracrine function of CMECs is regulated by MIRI and resveratrol remains to be elucidated. CMECs pretreated with/ without resveratrol were subjected to hypoxia/reoxygenation (H/R). Apoptosis was measured by flowcytometry. Protein antibody arrays were performed to find the alteration of cytokine secreted by CMECs. The Gene Ontology analysis was applied to interpret the function of modulated factors. We revealed resveratrol inhibited apoptosis of CMECs dose-dependently after H/R and reached its peak effect at the concentration of 100 µM. 29 factors were significantly changed by H/R, and resveratrol at 100 µM changed 98 types of factors compared with the H/R group. Among these factors, eight were increased by H/R and then were decreased by resveratrol. Eleven were attenuated by H/R and further decreased by resveratrol. Insulin-like growth factor binding protein-1 was upregulated by H/R and it was further increased by resveratrol. The altered factors were involved in cell proliferation, cell growth, cell motility, chemotaxis, angiogenesis and vasculogenesis. The study suggests that resveratrol inhibits the apoptosis and modulates the paracrine function of CMECs under ischemia/reperfusion condition.

Wellens' syndrome: incidence, characteristics, and long-term clinical outcomes.

BACKGROUND: Few studies with large sample sizes are available regarding patients with Wellens' syndrome. Therefore, we sought to assess the current incidence, risk factors, clinical presentation and long-term outcomes of this population. METHODS: Among a total of 3528 patients with ACS who underwent angioplasty from 2017 to 2019 in our centre, 2127 NSTE-ACS patients with culprit LAD vessels were enrolled in this study. According to electrocardiographic criteria, the patients were divided into a Wellens' group (n = 200) and non-Wellens' group (n = 1927). The primary endpoint was cardiac death; the secondary endpoint was MACCE, a composite of all-cause death, cardiac death, recurrent myocardial infarction, target lesion revascularization, heart failure and stroke. RESULTS: The incidence of Wellens' syndrome was 5.7% (200 of 3528) of all ACS patients. Wellens' syndrome more often manifested as NSTEMI (69% vs. 17.5%, P < 0.001). The percentages of preexisting coronary heart disease (39.6% vs. 23%) and previous PCI (19.5% vs. 9%) were significantly higher in the non-Wellens' group than in the Wellens' group (all P < 0.001). More importantly, the proportion of early PCI was higher in the Wellens' group (68% vs. 59.3%, P = 0.017). At a median follow-up of 24 months, Wellens' syndrome was not associated with an increased risk of MACCE (P = 0.05) or cardiac death (P = 0.188). CONCLUSIONS: The presence of Wellens' syndrome is not definitively associated with adverse prognosis in patients with NSTE-ACS. Age ≥ 65 years, diabetes, NSTEMI, eGFR < 60 ml/min and left main disease are associated with the incidence of cardiac death. Early recognition and aggressive intervention are critical, as they may help to attenuate adverse outcomes.

Planning CT-guided robust and fast cone-beam CT scatter correction using a local filtration technique.

PURPOSE: Cone-beam CT (CBCT) has been widely utilized in image-guided radiotherapy. Planning CT (pCT)-aided CBCT scatter correction could further enhance image quality and extend CBCT application to dose calculation and adaptive planning. Nevertheless, existing pCT-based approaches demand accurate registration between pCT and CBCT, leading to limited imaging performance and increased computational cost when large anatomical discrepancies exist. In this work, we proposed a robust and fast CBCT scatter correction method using local filtration technique and rigid registration between pCT and CBCT (LF-RR). METHODS: First of all, the pCT was rigidly registered with CBCT, then forward projection was performed on registered pCT to create scatter-free projections. The raw scatter signals were obtained via subtracting the scatter-free projections from the measured CBCT projections. Based on frequency and intensity threshold criteria, reliable scatter signals were selected from the raw scatter signals, and further filtered for global scatter estimation via local filtration technique. Finally, corrected CBCT was reconstructed with the projections generated by subtracting the scatter estimation from the raw CBCT projections using FDK algorithm. The LF-RR method was evaluated via comparison with another pCT-based scatter correction method based on Median and Gaussian filters (MG method). RESULTS: Proposed method was first validated on an anthropomorphic pelvis phantom, and showed satisfied performance on scatter removal even when anatomical mismatches were intentionally created on pCT. The quantitative analysis was further performed on four clinical CBCT images. Compared with the uncorrected CBCT, CBCT corrected by MG with rigid registration (MG-RR), MG with deformable registration (MG-DR), and LF-RR reduced the CT number error from 79 ± 35 to 25 ± 18 , 17 ± 13 and 7 ± 3 HU for adipose and from 115 ± 61 to 36 ± 22 , 30 ± 24 , 7 ± 3 HU for muscle, respectively. After correction, the spatial non-uniformity (SNU) of CBCT corrected with MG-RR, MG-DR and LF-RR was 51 ± 13 , 60 ± 21 , and 21 ± 9 HU for adipose, and 50 ± 22 , 57 ± 41 , and 25 ± 6 HU for muscle, respectively. Meanwhile, the contrast-to-noise ratio (CNR) between muscle and adipose was increased by a factor of 2.70, 2.89 and 2.56, respectively. Using the LF-RR method, the scatter correction of 656 projections can be finished within 10 s and the corrected volumetric images (200 slices) can be obtained within 2 min. CONCLUSION: We developed a fast and robust pCT-based CBCT scatter correction method which exploits the local-filtration technique to promote the accuracy of scatter estimation and is resistant to pCT-to-CBCT registration uncertainties. Both phantom and patient studies showed the superiority of the proposed correction in imaging accuracy and computational efficiency, indicating promisingfuture clinical application.

Fast and effective single-scan dual-energy cone-beam CT reconstruction and decomposition denoising based on dual-energy vectorization.

PURPOSE: Flat-panel detector (FPD) based dual-energy cone-beam computed tomography (DE-CBCT) is a promising imaging technique for dedicated clinical applications. In this paper, we proposed a fully analytical method for fast and effective single-scan DE-CBCT image reconstruction and decomposition. METHODS: A rotatable Mo filter was inserted between an x-ray source and imaged object to alternately produce low and high-energy x-ray spectra. First, filtered-backprojection (FBP) method was applied on down-sampled projections to reconstruct low and high-energy images. Then, the two images were converted into a vectorized form represented with an amplitude and an argument image. Using amplitude image as a guide, a joint bilateral filter was applied to denoise the argument image. Then, high-quality dual-energy images were recovered from the amplitude image and the denoised argument image. Finally, the recovered dual-energy images were further used for low-noise material decomposition and electron density synthesis. Imaging was conducted on a Catphan® 600 phantom and an anthropomorphic head phantom. The proposed method was evaluated via comparison with the traditional two-scan method and a commonly used filtering method (HYPR-LR). RESULTS: On the Catphan® 600 phantom, the proposed method successfully reduced streaking artifacts and preserved spatial resolution and noise-power-spectrum (NPS) pattern. In the electron density image, the proposed method increased contrast-to-noise ratio (CNR) by more than 2.5 times and achieved <1.2% error for electron density values. On the anthropomorphic head phantom, the proposed method greatly improved the soft-tissue contrast and the fine detail differentiation ability. In the selected ROIs on different human tissues, the differences between the CT number obtained by the proposed method and that by the two-scan method were less than 4 HU. In the material images, the proposed method suppressed noise by over 75.5% compared with two-scan results, and by over 40.4% compared with HYPR-LR results. Implementation of the whole algorithm took 44.5 s for volumetric imaging, including projection preprocessing, FBP reconstruction, joint bilateral filtering, and material decomposition. CONCLUSIONS: Using down-sampled projections in single-scan DE-CBCT, the proposed method could effectively and efficiently produce high-quality DE-CBCT images and low-noise material decomposition images. This method demonstrated superior performance on spatial resolution enhancement, NPS preservation, noise reduction, and electron density accuracy, indicating better prospect in material differentiation and dose calculation.

Urinary Alpha1-Microglobulin: A New Predictor for In-Hospital Mortality in Patients with ST-Segment Elevation Myocardial Infarction.

BACKGROUND Alpha1-microglobulin (A1MG) is a small molecular protein related to oxidation and inflammation. It exists in diverse body fluids, including urine. Results from urine tests are sometimes neglected when predicting in-hospital prognosis. It remains unclear whether urinary A1MG (UA1MG) can predict short-term prognosis of ST-elevated myocardial infarction (STEMI). MATERIAL AND METHODS A total of 1854 hospitalized patients with acute STEMI were retrospectively enrolled in our study. Medical records were used to obtain patient demographic and clinical information, UA1MG values (which were used to divide patients into groups of low, medium, or high), and other laboratory parameters. Principal clinical outcomes of interest were all-cause in-hospital deaths, cardiac deaths, and major adverse cardiac events (MACEs). RESULTS Among the 1854 enrolled patients, 43 (2.3%) died in the hospital, of which 33 (1.8%) were cardiac deaths. MACEs were noted in 113 patients (6.1%) during hospitalization. The group with the highest UA1MG value showed a significantly higher frequency of in-hospital deaths, cardiac deaths, and MACEs, compared to those of the lowest UA1MG value group (4.4% vs. 1.0%, P<0.001; 3.1% vs. 0.6%, P<0.005; and 8.6% vs. 4.7%, P=0.007, respectively). Multivariate regression analysis revealed that UA1MG levels (odds ratio 1.109, 95% confidence interval (CI) 1.027-1.197, P=0.008) independently predicted all-cause in-hospital mortality. A UA1MG value of 3.23 mg/dL was considered as an optimal cutoff point in STEMI to predict all-cause mortality after receiver operating characteristic curve analysis (area under the curve 0.73, 95% CI 0.65-0.80, P<0.001). CONCLUSIONS The UA1MG value at hospital admission could be an independent prognostic factor of all-cause in-hospital mortality in patients with STEMI.

Tongxinluo attenuates oxygen-glucose-serum deprivation/restoration-induced endothelial barrier breakdown via peroxisome proliferator activated receptor-α/angiopoietin-like 4 pathway in high glucose-incubated human cardiac microvascular endothelial cells.

BACKGROUND: Traditional Chinese medicine Tongxinluo (TXL) has been widely used to treat coronary artery disease in China, since it could reduce myocardial infarct size and ischemia/reperfusion injury in both non-diabetic and diabetic conditions. It has been shown that TXL could regulate peroxisome proliferator activated receptor-α (PPAR-α), a positive modulator of angiopoietin-like 4 (Angptl4), in diabetic rats. Endothelial junction substructure components, such as VE-cadherin, are involved in the protection of reperfusion injury. Thus, we hypothesized cell-intrinsic and endothelial-specific Angptl4 mediated the protection of TXL on endothelial barrier under high glucose condition against ischemia/reperfusion-injury via PPAR-α pathway. METHODS: Incubated with high glucose medium, the human cardiac microvascular endothelial cells (HCMECs) were then exposed to oxygen-glucose-serum deprivation (2 hours) and restoration (2 hours) stimulation, with or without TXL, insulin, or rhAngptl4 pretreatment. RESULTS: TXL, insulin, and rhAngptl4 had similar protective effects on the endothelial barrier. TXL treatment reversed the endothelial barrier breakdown in HCMECs significantly as identified by decreasing endothelial permeability, upregulating the expression of JAM-A, VE-cadherin, and integrin-α5 and increasing the membrane location of VE-cadherin and integrin-α5, and these effects of TXL were as effective as insulin and rhAngptl4. However, Angptl4 knock-down with small interfering RNA (siRNA) interference and PPAR-α inhibitor MK886 partially abrogated these beneficial effects of TXL. Western blotting also revealed that similar with insulin, TXL upregulated the expression of Angptl4 in HCMECs, which could be inhibited by Angptl4 siRNA or MK886 exposure. TXL treatment increased PPAR-α activity, which could be diminished by MK886 but not by Angptl4 siRNA. CONCLUSION: These data suggest cell-intrinsic and endothelial-specific Angptl4 mediates the protection of TXL against endothelial barrier breakdown during oxygen-glucose-serum deprivation and restoration under high glucose condition partly via the PPAR-α/Angptl4 pathway.

The Neutrophil Percentage to Albumin Ratio as a New Predictor of In-Hospital Mortality in Patients with ST-Segment Elevation Myocardial Infarction.

BACKGROUND Neutrophil and albumin are respective indicators of inflammation and malnutrition. Whether combining those 2 markers can predict acute prognosis in patients with ST-segment elevation myocardial infarction (STEMI) remains unknown. This study aimed to investigate the prognostic value of neutrophil percentage to albumin ratio (NPAR) for in-hospital mortality in STEMI patients. MATERIAL AND METHODS There were 1024 patients hospitalized with acute STEMI retrospectively enrolled in this study. Demographic, clinical, and admission laboratory data were extracted from medical record. NPAR was calculated as neutrophil percentage numerator divided by albumin in the admission blood samples. In-hospital mortality was designed as the primary outcome in the study, major adverse cardiac events (MACE) and cardiac death were recorded as the secondary clinical outcomes. RESULTS The rates of in-hospital mortality, MACE, and cardiac death in high NPAR group were significantly higher than those in the low NPAR group (P<0.001, P=0.004, P<0.001). The Kaplan-Meier analysis showed worse outcomes in higher NPAR group (P<0.001). NPAR levels and age independently predicted in-hospital mortality. A NPAR value >1.9 was identified as an effective cut point in STEMI for in-hospital mortality (P<0.001, sensitivity 82%, specificity 52%). CONCLUSIONS Admission NPAR was independently correlated with in-hospital mortality in patients with STEMI.

Tongxinluo attenuates reperfusion injury in diabetic hearts by angiopoietin-like 4-mediated protection of endothelial barrier integrity via PPAR-α pathway.

OBJECTIVE: Endothelial barrier function in the onset and Tongxinluo (TXL) protection of myocardial ischemia/reperfusion (I/R) injury, and TXL can induce the secretion of Angiopoietin-like 4 (Angptl4) in human cardiac microvascular endothelial cells during hypoxia/reoxygenation. We intend to demonstrate whether TXL can attenuate myocardial I/R injury in diabetes, characterized with microvascular endothelial barrier disruption, by induction of Angptl4-mediated protection of endothelial barrier integrity. METHODS AND RESULTS: I/R injury was created by coronary ligation in ZDF diabetic and non-diabetic control rats. The animals were anesthetized and randomized to sham operation or I/R injury with or without the exposure to insulin, rhAngptl4, TXL, Angptl4 siRNA, and the PPAR-α inhibitor MK886. Tongxinluo, insulin and rhAngptl4 have the similar protective effect on diabetic hearts against I/R injury. In I/R-injured diabetic hearts, TXL treatment remarkably reduced the infarct size, and protected endothelial barrier integrity demonstrated by decreased endothelial cells apoptosis, microvascular permeability, and myocardial hemorrhage, fortified tight junction, and upregulated expression of JAM-A, integrin-α5, and VE-cadherin, and these effects of TXL were as effective as insulin and rhAngptl4. However, Angptl4 knock-down with siRNA interference and inhibition of PPAR-α with MK886 partially diminished these beneficial effects of TXL and rhAngptl4. TXL induced the expression of Angptl4 in I/R-injured diabetic hearts, and was canceled by Angptl4 siRNA and MK886. TXL treatment increased myocardial PPAR-α activity, and was abolished by MK886 but not by Angptl4 siRNA. CONCLUSIONS: TXL protects diabetic hearts against I/R injury by activating Angptl4-mediated restoration of endothelial barrier integrity via the PPAR-α pathway.

Inhibition of miR-128-3p by Tongxinluo Protects Human Cardiomyocytes from Ischemia/reperfusion Injury via Upregulation of p70s6k1/p-p70s6k1.

Background and Aims: Tongxinluo (TXL) is a multifunctional traditional Chinese medicine that has been widely used to treat cardiovascular and cerebrovascular diseases. However, no studies have explored whether TXL can protect human cardiomyocytes (HCMs) from ischemia/reperfusion (I/R) injury. Reperfusion Injury Salvage Kinase (RISK) pathway activation was previously demonstrated to protect the hearts against I/R injury and it is generally activated via Akt or (and) Erk 1/2, and their common downstream protein, ribosomal protein S6 kinase (p70s6k). In addition, prior studies proved that TXL treatment of cells promoted secretion of VEGF, which could be stimulated by the increased phosphorylation of one p70s6k subtype, p70s6k1. Consequently, we hypothesized TXL could protect HCMs from I/R injury by activating p70s6k1 and investigated the underlying mechanism. Methods and Results: HCMs were exposed to hypoxia (18 h) and reoxygenation (2 h) (H/R), with or without TXL pretreatment. H/R reduced mitochondrial membrane potential, increased bax/bcl-2 ratios and cytochrome C levels and induced HCM apoptosis. TXL preconditioning reversed these H/R-induced changes in a dose-dependent manner and was most effective at 400 µg/mL. The anti-apoptotic effect of TXL was abrogated by rapamycin, an inhibitor of p70s6k. However, inhibitors of Erk1/2 (U0126) or Akt (LY294002) failed to inhibit the protective effect of TXL. TXL increased p70s6k1 expression and, thus, enhanced its phosphorylation. Furthermore, transfection of cardiomyocytes with siRNA to p70s6k1 abolished the protective effects of TXL. Among the micro-RNAs (miR-145-5p, miR-128-3p and miR-497-5p) previously reported to target p70s6k1, TXL downregulated miR-128-3p in HCMs during H/R, but had no effects on miR-145-5p and miR-497-5p. An in vivo study confirmed the role of the p70s6k1 pathway in the infarct-sparing effect of TXL, demonstrating that TXL decreased miR-128-3p levels in the rat myocardium during I/R. Transfection of HCMs with a hsa-miR-128-3p mimic eliminated the protective effects of TXL. Conclusions: The miR-128-3p/p70s6k1 signaling pathway is involved in protection by TXL against HCM apoptosis during H/R. Overexpression of p70s6k1 is, therefore, a potential new strategy for alleviating myocardial reperfusion injury.

Quantitative Proteomics Analysis of Ischemia/Reperfusion Injury-Modulated Proteins in Cardiac Microvascular Endothelial Cells and the Protective Role of Tongxinluo.

BACKGROUND: The protection of endothelial cells (ECs) against reperfusion injury has received little attention. In this study, we used Tandem Mass Tag (TMT) labeling proteomics to investigate the modulated proteins in an in vitro model of cardiac microvascular endothelial cells (CMECs) subjected to ischemia/reperfusion (I/R) injury and their alteration by traditional Chinese medicine Tongxinluo (TXL). METHODS: Human CMECs were subjected to 2 h of hypoxia followed by 2 h of reoxygenation with different concentrations of TXL Protein expression profiles of CMECs were determined using tandem mass spectrometry. We evaluated several proteins with altered expression in I/R injury and summarized some reported proteins related to I/R injury. RESULTS: TXL dose-dependently decreased CMEC apoptosis, and the optimal concentration was 800 µg/mL. I/R significantly altered proteins in CMECs, and 30 different proteins were detected between a normal group and a hypoxia and serum deprivation group. In I/R injury, TXL treatment up-regulated 6 types of proteins including acyl-coenzyme A synthetase ACSM2B mitochondrial (ACSM2B), cyclin-dependent kinase inhibitor 1B (CDKN1B), heme oxygenase 1 (HMOX1), transcription factor SOX-17 (SOX17), sequestosome-1 isoform 1 (SQSTM1), and TBC1 domain family member 10B (TBC1D10B). Also, TXL down-regulated 5 proteins including angiopoietin-2 isoform c precursor (ANGPT2), cytochrome c oxidase assembly factor 5 (COA5), connective tissue growth factor precursor (CTGF), cathepsin L1 isoform 2 (CTSL), and eukaryotic elongation factor 2 kinase (LOC101930123). These types of proteins mainly had vital functions, including cell proliferation, stress response, and regulation of metabolic process. CONCLUSIONS: The study presented differential proteins upon I/R injury through a proteomic analysis. TXL modulated the expression of proteins in CMECs and has a protective role in response to I/R.

Tongxinluo exerts protective effects via anti-apoptotic and pro-autophagic mechanisms by activating AMPK pathway in infarcted rat hearts.

NEW FINDINGS: What is the central question of this study? In a rat model of acute myocardial infarction (AMI), we investigated the effect of Tongxinluo (TXL) treatment. Does TXL activate autophagy and attenuate apoptosis of cardiomyocytes through the AMPK pathway to facilitate survival of cardiomyocytes and improve cardiac function? What is the main finding and its importance? Major findings are as follows: (i) TXL treatment preserved cardiac function and reduced ventricular remodelling, infarct size and inflammation in rat hearts after AMI; (ii) TXL treatment dramatically increased autophagy and inhibited apoptosis in myocardium; and (iii) the AMPK signalling pathway played a crucial role in mediating the beneficial effects of TXL. Tongxinluo (TXL) has been demonstrated to have a protective role during ischaemia-reperfusion after acute myocardial infarction, but the long-term effects and underlying mechanisms are still unknown. The aim of this study was to investigate whether TXL could have an effect on apoptosis or autophagy of cardiomyocytes through the AMP-activated protein kinase (AMPK) pathway. Male Sprague-Dawley rats (n = 75) were randomly divided to sham, control, TXL (4 mg kg-1  day-1 orally), compound C (i.p. injection of 10 mg kg-1  day-1 ) and TXL + compound C groups. The extent of fibrosis, infarct size and angiogenesis were determined by pathological and histological studies. Four weeks after acute myocardial infarction, TXL treatment significantly increased ejection fraction, promoted angiogenesis in the peri-infarct region and substantially decreased fibrosis and the size of the infarcted area (P < 0.05). Treatment with TXL also increased AMPK/mTOR phosphorylation, upregulated expression of the autophagic protein LC3 and downregulated expression of the apoptotic protein Bax in the infarcted myocardium (P < 0.05). Addition of the AMPK inhibitor, compound C, counteracted these beneficial effects significantly (P < 0.05). The cardioprotective benefits of TXL against myocardial infarction are related to the inhibition of apoptosis and promotion of autophagy in rat hearts after acute myocardial infarction. This effect may occur through the AMPK signalling pathway.

Tongxinluo modulates cytokine secretion by cardiac microvascular endothelial cells in ischemia/reperfusion injury.

Cardiac microvascular endothelial cells (CMECs) extensively secrete cytokines during myocardial ischemia/reperfusion injury (MIRI). Tongxinluo (TXL) has been demonstrated to preserve the function of the endothelium and myocardium against MIRI. This study was designed to identify alterations in the paracrine function of CMECs under hypoxia/reoxygenation (H/R) conditions and assess its modulation by TXL. CMECs were exposed to different concentrations of TXL for 30 min and then subjected to hypoxia and reoxygenation for 12 and 2 h, respectively. Apoptosis was measured to determine the optimal TXL concentration. Protein antibody arrays were used to assess changes in cytokines secreted into conditioned medium by CMECs. A Gene Ontology (GO) analysis was applied to interpret the functional implications of changes in cytokines. TXL inhibited CMEC apoptosis in a concentration-dependent manner after H/R, reaching peak efficacy at a concentration of 800 µg/ml. H/R significantly altered 33 cytokines, and TXL (800 µg/ml) changed the levels of 121 different cytokines compared with the H/R group. Among these cytokines, 10 that were increased by H/R were decreased by TXL, five that were decreased by H/R were increased by TXL, and eight that were attenuated by H/R were further decreased by TXL. Insulin-like growth factor binding protein-1 was up-regulated by H/R and was further increased by TXL. Significantly altered factors were found to be involved in cell proliferation, growth and differentiation, as well as chemotaxis and transport. TXL inhibited the apoptosis of CMECs and modulated their paracrine function in MIRI.

Cardiac microvascular barrier function mediates the protection of Tongxinluo against myocardial ischemia/reperfusion injury.

OBJECTIVE: Tongxinluo (TXL) has been shown to decrease myocardial necrosis after ischemia/reperfusion (I/R) by simulating ischemia preconditioning (IPC). However, the core mechanism of TXL remains unclear. This study was designed to investigate the key targets of TXL against I/R injury (IRI) among the cardiac structure-function network. MATERIALS AND METHODS: To evaluate the severity of lethal IRI, a mathematical model was established according to the relationship between myocardial no-reflow size and necrosis size. A total of 168 mini-swine were employed in myocardial I/R experiment. IRI severity among different interventions was compared and IPC and CCB groups were identified as the mildest and severest groups, respectively. Principal component analysis was applied to further determine 9 key targets of IPC in cardioprotection. Then, the key targets of TXL in cardioprotection were confirmed. RESULTS: Necrosis size and no-reflow size fit well with the Sigmoid Emax model. Necrosis reduction space (NRS) positively correlates with I/R injury severity and necrosis size (R2=0.92, R2=0.57, P<0.01, respectively). Functional and structural indices correlate positively with NRS (R2=0.64, R2=0.62, P<0.01, respectively). TXL recovers SUR2, iNOS activity, eNOS activity, VE-cadherin, ß-catenin, γ-catenin and P-selectin with a trend toward the sham group. Moreover, TXL increases PKA activity and eNOS expression with a trend away from the sham group. Among the above nine indices, eNOS activity, eNOS, VE-cadherin, ß-catenin and γ-catenin expression were significantly up-regulated by TXL compared with IPC (P>0.05) or CCB (P<0.05) and these five microvascular barrier-related indices may be the key targets of TXL in minimizing IRI. CONCLUSIONS: Our study underlines the lethal IRI as one of the causes of myocardial necrosis. Pretreatment with TXL ameliorates myocardial IRI through promoting cardiac microvascular endothelial barrier function by simulating IPC.

Induction of autophagy by Tongxinluo through the MEK/ERK pathway protects human cardiac microvascular endothelial cells from hypoxia/reoxygenation injury.

: In contrast to cardiomyocytes, autophagy in cardiac microvascular endothelial cells (CMECs) during ischemia/reperfusion (I/R) injury has not been fully investigated. Tongxinluo (TXL), a traditional Chinese medicine, was shown to be vascular protective. We aimed to elucidate the role of autophagy and its regulatory mechanisms by TXL in CMECs subjected to I/R injury. CMECs were exposed to different treatments for 30 minutes and subjected to hypoxia/reoxygenation each for 2 hours. The results indicated that hypoxia/reoxygenation significantly induced autophagy, as identified by an increased number of monodansylcadaverine-positive CMECs, increased autophagosome formation, and a higher type II/type I of light chain 3 ratio, but not Beclin-1 expression. Autophagy inhibition using 3-methyladenine was proapoptotic, but rapamycin-induced autophagy was antiapoptotic. TXL enhanced autophagy and decreased apoptosis in a dose-dependent manner, reaching its largest effect at 800 µg/mL. 3-methyladenine attenuated the TXL-promoted autophagy and antiapoptotic effects, whereas rapamycin had no additional effects compared with TXL alone. TXL upregulated mitogen-activated protein kinase and extracellular signal-regulated kinase (ERK) phosphorylation; however, PD98059 abrogated ERK phosphorylation and decreased autophagy and increased apoptosis compared with TXL alone. These results suggest that autophagy is a protective mechanism in CMECs subjected to I/R injury and that TXL can promote autophagy through activation of the mitogen-activated protein kinase/ERK pathway.