Endothelial YAP Mediates Hyperglycemia-Induced Platelet Hyperactivity and Arterial Thrombosis.

BACKGROUND: Hyperglycemia-a symptom that characterizes diabetes-is highly associated with atherothrombotic complications. However, the underlying mechanism by which hyperglycemia fuels platelet activation and arterial thrombus formation is still not fully understood. METHODS: The profiles of polyunsaturated fatty acid metabolites in the plasma of patients with diabetes and healthy controls were determined with targeted metabolomics. FeCl3-induced carotid injury model was used to assess arterial thrombus formation in mice with endothelial cell (EC)-specific YAP (yes-associated protein) deletion or overexpression. Flow cytometry and clot retraction assay were used to evaluate platelet activation. RNA sequencing and multiple biochemical analyses were conducted to unravel the underlying mechanism. RESULTS: The plasma PGE2 (prostaglandin E2) concentration was elevated in patients with diabetes with thrombotic complications and positively correlated with platelet activation. The PGE2 synthetases COX-2 (cyclooxygenase-2) and mPGES-1 (microsomal prostaglandin E synthase-1) were found to be highly expressed in ECs but not in other type of vessel cells in arteries from both patients with diabetes and hyperglycemic mice, compared with nondiabetic individuals and control mice, respectively. A combination of RNA sequencing and ingenuity pathway analyses indicated the involvement of YAP signaling. EC-specific deletion of YAP limited platelet activation and arterial thrombosis in hyperglycemic mice, whereas EC-specific overexpression of YAP in mice mimicked the prothrombotic state of diabetes, without affecting hemostasis. Mechanistically, we found that hyperglycemia/high glucose-induced endothelial YAP nuclear translocation and subsequently transcriptional expression of COX-2 and mPGES-1 contributed to the elevation of PGE2 and platelet activation. Blockade of EP3 (prostaglandin E receptor 3) activation by oral administration of DG-041 reversed the hyperactivity of platelets and delayed thrombus formation in both EC-specific YAP-overexpressing and hyperglycemic mice. CONCLUSIONS: Collectively, our data suggest that hyperglycemia-induced endothelial YAP activation aggravates platelet activation and arterial thrombus formation via PGE2/EP3 signaling. Targeting EP3 with DG-041 might be therapeutic for diabetes-related thrombosis.

Splenic monocytes mediate inflammatory response and exacerbate myocardial ischemia/reperfusion injury in a mitochondrial cell-free DNA-TLR9-NLRP3-dependent fashion.

The spleen contributes importantly to myocardial ischemia/reperfusion (MI/R) injury. Nucleotide-binding oligomerization domain-like receptor family pyrin domain containing 3 (NLRP3) recruits inflammasomes, initiating inflammatory responses and mediating tissue injury. We hypothesize that myocardial cell-free DNA (cfDNA) activates the splenic NLRP3 inflammasome during early reperfusion, increases systemic inflammatory response, and exacerbates myocardial infarct. Mice were subjected to 40 min of ischemia followed by 0, 1, 5, or 15 min, or 24 h of reperfusion. Splenic leukocyte adoptive transfer was performed by injecting isolated splenocytes to mice with splenectomy performed prior to left coronary artery occlusion. CY-09 (4 mg/kg) was administered 5 min before reperfusion. During post-ischemic reperfusion, splenic protein levels of NLRP3, cleaved caspase-1, and interleukin-1ß (IL-1ß) were significantly elevated and peaked (2.1 ± 0.2-, 3.4 ± 0.4-, and 3.2 ± 0.2-fold increase respectively, p < 0.05) within 5 min of reperfusion. In myocardial tissue, NLRP3 was not upregulated until 24 h after reperfusion. Suppression by CY09, a specific NLRP3 inflammasome inhibitor, or deficiency of NLRP3 significantly reduced myocardial infarct size (17.3% ± 4.2% and 33.2% ± 1.8% decrease respectively, p < 0.01). Adoptive transfer of NLRP3-/- splenocytes to WT mice significantly decreased infarct size compared to transfer of WT splenocytes (19.1% ± 2.8% decrease, p < 0.0001). NLRP3 was mainly activated at 5 min after reperfusion in CD11b+ and LY6G- splenocytes, which significantly increased during reperfusion (24.8% ± 0.7% vs.14.3% ± 0.6%, p < 0.0001). The circulating cfDNA level significantly increased in patients undergoing cardiopulmonary bypass (CPB) (43.3 ± 5.3 ng/mL, compared to pre-CPB 23.8 ± 3.5 ng/mL, p < 0.01). Mitochondrial cfDNA (mt-cfDNA) contributed to NLRP3 activation in macrophages (2.1 ± 0.2-fold increase, p < 0.01), which was inhibited by a Toll-like receptor 9(TLR9) inhibitor. The NLRP3 inflammasome in splenic monocytes is activated and mediates the inflammatory response shortly after reperfusion onset, exacerbating MI/R injury in mt-cfDNA/TLR9-dependent fashion. The schema reveals splenic NLRP3 mediates the inflammatory response in macrophages and exacerbates MI/R in a mitochondrial cfDNA/ TLR9-dependent fashion.

Association between fluid balance and mortality for heart failure and sepsis: a propensity score-matching analysis.

BACKGROUND: Fluid resuscitation is necessary to correct the sepsis-induced hypoperfusion, which is contradictory to the treatment of heart failure. This study explored the association between fluid balance (FB) of the first 24 h after ICU admission and mortality in critically ill patients with heart failure and sepsis. METHODS: Data were extracted from the Medical Information Mart for Intensive Care database. The locally weighted scatterplot smoothing (Lowess) method was used to demonstrate the relationship between FB and in-hospital mortality. Groups were divided into high FB (≥ 55.85 ml/kg) and low FB (< 55.85 ml/kg) according to the cut-off value of FB using Receiver operating characteristic analysis and Youden index method. The primary outcome was in-hospital mortality. Subgroup analyses, multivariable logistic regression analyses, and Kaplan-Meier curves were used to detect the association and survival difference between groups. Inverse probability treatment weighting (IPTW) and propensity score matching (PSM) were performed to minimize the bias of confounding factors and facilitate the comparability between groups. RESULTS: A total of 936 patients were included. The Lowess curve showed an approximate positive linear relationship for FB and in-hospital mortality. In the multivariable logistic regression adjusted model, high FB showed strong associations with in-hospital mortality (OR 2.53, 95% CI 1.60-3.99, p < 0.001) as compared to the low FB group. In IPTW and PSM models, high FB consistently showed higher in-hospital mortality (IPTW model: OR 1.94, 95% CI 1.52-2.49, p < 0.001; PSM model: OR 2.93, 95% CI 1.75-4.90, p < 0.001) and 30-day mortality (IPTW model: OR 1.65, 95% CI 1.29-2.10, p < 0.001; PSM model: OR 2.50, 95% CI 1.51-4.15, p < 0.001), compared with the low FB group. CONCLUSION: For critically ill patients with heart failure and sepsis, high FB within the first 24 h after ICU admission could serve as an independent risk factor for in-hospital mortality and 30-day mortality. The avoidance of fluid overload exerts important effects on reducing mortality in such patients.

Histone demethylase KDM3C regulates the lncRNA GAS5-miR-495-3p-PHF8 axis in cardiac hypertrophy.

Cardiac hypertrophy (CH) is a pathological phenotype of cardiomyopathy. Epigenetic modification is a mechanism associated with CH. Our study here investigated the histone demethylase KDM3C in relation to epigenetic regulation in CH. We found that KDM3C mRNA silencing alleviated CH, as evidenced by reduced ANP, BNP, and ß-MHC mRNAs, increased α-MHC mRNA, decreased cell surface area, and reduced cellular protein/DNA ratios. Specifically, KDM3C upregulated miR-200c-3p expression through demethylation of H3K9me2, leading to enhanced binding of miR-200c-3p to GAS5 and suppression of GAS5 expression; these effects then led to reduced binding of GAS5 to miR-495-3p, increased miR-495-3p expression, and repression of PHF8 transcription. Through these mechanisms, our data indicate that KDM3C-dependent epigenetic modification promotes CH.

Disruption of USP9X in macrophages promotes foam cell formation and atherosclerosis.

Subendothelial macrophage internalization of modified lipids and foam cell formation are hallmarks of atherosclerosis. Deubiquitinating enzymes (DUBs) are involved in various cellular activities; however, their role in foam cell formation is not fully understood. Here, using a loss-of-function lipid accumulation screening, we identified ubiquitin-specific peptidase 9 X-linked (USP9X) as a factor that suppressed lipid uptake in macrophages. We found that USP9X expression in lesional macrophages was reduced during atherosclerosis development in both humans and rodents. Atherosclerotic lesions from macrophage USP9X-deficient mice showed increased macrophage infiltration, lipid deposition, and necrotic core content than control apolipoprotein E-KO (Apoe-/-) mice. Additionally, loss-of-function USP9X exacerbated lipid uptake, foam cell formation, and inflammatory responses in macrophages. Mechanistically, the class A1 scavenger receptor (SR-A1) was identified as a USP9X substrate that removed the K63 polyubiquitin chain at the K27 site. Genetic or pharmacological inhibition of USP9X increased SR-A1 cell surface internalization after binding of oxidized LDL (ox-LDL). The K27R mutation of SR-A1 dramatically attenuated basal and USP9X knockdown-induced ox-LDL uptake. Moreover, blocking binding of USP9X to SR-A1 with a cell-penetrating peptide exacerbated foam cell formation and atherosclerosis. In this study, we identified macrophage USP9X as a beneficial regulator of atherosclerosis and revealed the specific mechanisms for the development of potential therapeutic strategies for atherosclerosis.

Nrf2 participates in the protective effect of exogenous mitochondria against mitochondrial dysfunction in myocardial ischaemic and hypoxic injury.

OBJECTIVE: Coronary artery disease is one of the leading causes of death worldwide. Treatments including coronary artery intervention can cause complications, such as myocardial ischaemia-reperfusion injury (MIRI). Mitochondrial injury or dysfunction is a key pathology of MIRI. Mitochondrial transplantation is considered a promising therapeutic strategy for cardiac-related diseases, but its mechanism is still unclear. Nrf2 is a prominent player in supporting the structural and functional integrity of mitochondria. In our research, we focused on the effect of Nrf2 in the treatment of MIRI by mitochondrial transplantation. H9C2 cells were subjected to hypoxia/reoxygenation (H/R) and MIRI was induced in wild-type and Nrf2-/- mice by surgical ligation of the left coronary artery to elucidate the mechanism in vitro and in vivo, respectively. Exogenous mitochondria were extracted from healthy H9C2 cells and the pectoralis major and administered to H9C2 cells and mice with MIRI, respectively. Mitochondrial internalization, H9C2 cell injury or apoptosis, cardiac injury/function, mitochondrial function, morphology, mitochondrial dynamics, and the expression of components of the Nrf2 pathway were assessed. We found that exogenous mitochondria were internalized into H9C2 cardiomyocytes. Exogenous mitochondrial transplantation attenuated cardiomyocyte injury, cardiomyocyte apoptosis, and mitochondrial dysfunction. Exogenous mitochondrial transplantation increased the expression of Nrf2 and its downstream targets, attenuated cardiomyocyte injury, cardiac dysfunction, apoptosis, mitochondrial dysfunction, and mitochondrial fusion and fission imbalance, and improved mitophagy after MIRI in wild-type mice but not in Nrf2-/- mice. These results suggested that exogenous mitochondria can be internalized into cardiomyocytes and activate the Nrf2 pathway and that exogenous mitochondria improve cardiac function and ameliorate mitochondrial dysfunction via the Nrf2 pathway.

MiR-17-5p-mediated endoplasmic reticulum stress promotes acute myocardial ischemia injury through targeting Tsg101.

Cardiovascular diseases are the leading cause of death globally, among which acute myocardial infarction (AMI) frequently occurs in the heart and proceeds from myocardium ischemia and endoplasmic reticulum (ER) stress-induced cell death. Numerous studies on miRNAs indicated their potential as diagnostic biomarkers and treatment targets for heart diseases. Our study investigated the role of miR-17-5p and its regulatory mechanisms during AMI. Echocardiography, MTT, flow cytometry assay, evaluation of caspase-3 and lactate dehydrogenase (LDH) activity were conducted to assess cell viability, apoptosis in an MI/R mice model, and an H2O2-induced H9c2 hypoxia cell model, respectively. The expression levels of ER stress response-related biomarkers were detected using qRT-PCR, IHC, and western blotting assays. The binding site of miR-17-5p on Tsg101 mRNA was determined by bioinformatic prediction and luciferase reporter assay. The expression levels of miR-17-5p were notably elevated in MI/R mice and hypoxia cell models, accompanied by enhanced cell apoptosis. Inhibition of miR-17-5p led to decreased apoptosis related to ER stress response in the hypoxia model, which could be counteracted by knockdown of Tsg101 (tumor susceptibility gene 101). Transfection with miR-17-5p mimics downregulated the expression of Tsg101 in H9c2 cells. Luciferase assay demonstrated the binding between miR-17-5p and Tsg101. Moreover, 4-PBA, the inhibitor of the ER stress response, abolished shTsg101 elevated apoptosis in hypoxic H9c2 cells. Our findings investigated the pro-apoptotic role of miR-17-5p during MI/R, disclosed the specific mechanism of miR-17-5p/Tsg101 regulatory axis in ER stress-induced myocardium injury and cardiomyocytes apoptosis, and presented a promising diagnostic biomarker and potential target for therapy of AMI.

Long noncoding RNA MAGI1-IT1 regulates cardiac hypertrophy by modulating miR-302e/DKK1/Wnt/beta-catenin signaling pathway.

Cardiac hypertrophy (CH) is an adaptive cardiac response to overload whose decompensation eventually leads to heart failure or sudden death. Recently, accumulating studies have indicated the implication of long noncoding RNAs (lncRNAs) in CH progression. MAGI1-IT1 is a newly-identified lncRNA that is highly associated with CH, while its specific role in CH progression remains masked. In this study, we uncovered that MAGI1-IT1 was distinctly downregulated in angiotensin (Ang) II-induced hypertrophic H9c2 cells. Also, MAGI1-IT1 overexpression in Ang II-treated H9c2 cells strikingly abolished the enlarged surface area and the enhanced levels of hypertrophic markers such as ANP, BNP, and ß-MHC. Mechanically, we found MAGI1-IT1 sponged miR-302e which was identified as a hypertrophy-facilitator here, and that miR-302e upregulation countervailed the inhibition of MAGI1-IT1 overexpression on hypertrophic cells. Moreover, it was confirmed that MAGI1-IT1 boosted DKK1 expression by absorbing miR-302e. Subsequently, we also illustrated that MAGI1-IT1 inactivated Wnt/beta-catenin signaling through a DKK1-dependent pathway. Finally, both the DKK1 inhibition and LiCI (Wnt activator) supplement abrogated the hypertrophy-suppressive impact of MAGI1-IT1 on Ang II-simulated hypertrophic H9c2 cells. Jointly, our findings disclosed that MAGI1-IT1 functioned as a negative regulator in CH through inactivating Wnt/beta-catenin pathway via targeting miR-302e/DKK1 axis, revealing a novel road for CH treatment.

Heat shock protein 70 induced by heat stress protects heterotopically transplanted hearts in rats.

Heat shock protein 70 (HSP70) protects cardiac function against ischemia-reperfusion injury through gene transfection, although it is not a clinically practical and economical method. This study investigated whether heat stress-induced HSP70 protects heterotopically transplanted donor hearts. A total of 60 donor rats were randomly divided into 6 groups. Five of those received heat stress and one was a control group. Donor hearts were heterotopically transplanted into recipient rats at five time points, following the heat stress (0, 24, 48, 96 and 192 h). The levels of HSP70 expression in donor hearts and the variation of myocardial enzymes in receptor blood or donor hearts were measured 24 h after transplantation. The donated hearts were also examined under a microscope for pathological changes. HSP70 expression was the highest in the 24-h group (p≤0.01) and decreased gradually in the 48- and 96-h groups. No statistically significant difference was found in the HSP70 expression in the control, the 0- and 192-h groups (p≥0.05). Of all the groups, the 24-h group had the lowest lactate dehydrogenase and creatine kinase muscle band concentrations in receptor blood. Moreover, this group showed the lowest malondialdehyde concentration and the highest atriphosphate concentration (p≤0.01), demonstrated by the mildest inflammatory injury in the transplanted hearts. We found a time-dose-effect relationship among heat stress, HSP70 and the protection of donor hearts. Heat stress is a practical method that can be clinically applied to protect donor hearts against ischemia-reperfusion injury by inducing endogenous HSP70, which indicates the future direction of clinical practice.

Case control study of gastrointestinal complications after cardiopulmonary bypass heart surgery.

BACKGROUND: Gastrointestinal complications (GIC) after cardiopulmonary bypass (CPB) surgery are rare, but, nevertheless, extremely dangerous.The identification of risks for GIC may be helpful in planning appropriate perioperative management strategies. The aim of the present study was to analyze perioperative factors of GIC in patients undergoing CPB surgery. METHODS: We retrospectively analysed 206 patients who underwent GIC after cardiopulmonary bypass surgery from 2000 to 2007 and compared them with 206 matched control patients (matched for surgery, temperature, hemodilution and date). Univariate analysis and multiple logistic regression analysis were performed on 12 risk factors. RESULT: Sex and types of cardioplegia perfusate did not significantly influence the GIC after CPB surgery. Multiple logistic regression revealed that CPB time, preoperative serum creatinine (PSC) > or = 179 mg/dL, emergency surgery, perfusion pressure < or =40 mmHg, low cardiac output syndrome (LCOS), age > or = 61, mechanical ventilation > or =96 h, New York Heart Association (NYHA) class III and IV were predictors of the occurrence of GIC after CPB surgery. Perfusion pressure and aprotinin administration were protective factors. CONCLUSION: Gastrointestinal complications after CPB surgery could be predictive in the presence of the above risk factors. This study suggests that GIC can be reduced by maintenance of higher perfusion pressure and shortening the time on CPB and ventilation.

Evaluation of cardiac global function using the myocardial performance index by tissue Doppler echocardiography in patients with uremia.

OBJECTIVE: The purpose of this study was to assess global ventricular function in patients with uremia by means of the myocardial performance index (MPI) derived from tissue Doppler echocardiography. METHODS: According to the left ventricular mass index and pericardial effusion, 45 patients with uremia were classified into 2 groups: a uremia group and a uremia with pericardial effusion group. To calculate left ventricular MPI (LVMPI) and right ventricular MPI (RVMPI) by tissue Doppler echocardiography, the isovolumic contraction time (ICT), isovolumic relaxation time (IRT), and ejection time (ET) were measured at different sites in the mitral and tricuspid annuli. RESULTS: The mean ICT and IRT were longer, the ET was shorter, and the LVMPI and RVMPI were higher in the 2 disease groups than in a control group, and the indices were higher in the uremia with pericardial effusion group than in the uremia group. The increase of the LVMPI was more obvious than that of the RVMPI. There was a significant difference in the mean LVMPI and RVMPI among the 3 groups (P<.01). The MPI was positively correlated with the IRT and the sum of the ICT and IRT and negatively correlated with the ET. CONCLUSIONS: Both left and right ventricular systolic and diastolic function are impaired in patients with uremia. The MPI could be measured by tissue Doppler echocardiography, and we suggest that this index provides a novel, noninvasive method for clinical research on global myocardial performance in patients with uremia.