Endothelial extracellular vesicles induce acute lung injury via follistatin-like protein 1.

Cardiopulmonary bypass has been speculated to elicit systemic inflammation to initiate acute lung injury (ALI), including acute respiratory distress syndrome (ARDS), in patients after cardiac surgery. We previously found that post-operative patients showed an increase in endothelial cell-derived extracellular vesicles (eEVs) with components of coagulation and acute inflammatory responses. However, the mechanism underlying the onset of ALI owing to the release of eEVs after cardiopulmonary bypass, remains unclear. Plasma plasminogen-activated inhibitor-1 (PAI-1) and eEV levels were measured in patients with cardiopulmonary bypass. Endothelial cells and mice (C57BL/6, Toll-like receptor 4 knockout (TLR4-/-) and inducible nitric oxide synthase knockout (iNOS-/-)) were challenged with eEVs isolated from PAI-1-stimulated endothelial cells. Plasma PAI-1 and eEVs were remarkably enhanced after cardiopulmonary bypass. Plasma PAI-1 elevation was positively correlated with the increase in eEVs. The increase in plasma PAI-1 and eEV levels was associated with post-operative ARDS. The eEVs derived from PAI-1-stimulated endothelial cells could recognize TLR4 to stimulate a downstream signaling cascade identified as the Janus kinase 2/3 (JAK2/3)-signal transducer and activator of transcription 3 (STAT3)-interferon regulatory factor 1 (IRF-1) pathway, along with iNOS induction, and cytokine/chemokine production in vascular endothelial cells and C57BL/6 mice, ultimately contributing to ALI. ALI could be attenuated by JAK2/3 or STAT3 inhibitors (AG490 or S3I-201, respectively), and was relieved in TLR4-/- and iNOS-/- mice. eEVs activate the TLR4/JAK3/STAT3/IRF-1 signaling pathway to induce ALI/ARDS by delivering follistatin-like protein 1 (FSTL1), and FSTL1 knockdown in eEVs alleviates eEV-induced ALI/ARDS. Our data thus demonstrate that cardiopulmonary bypass may increase plasma PAI-1 levels to induce FSTL1-enriched eEVs, which target the TLR4-mediated JAK2/3/STAT3/IRF-1 signaling cascade and form a positive feedback loop, leading to ALI/ARDS after cardiac surgery. Our findings provide new insight into the molecular mechanisms and therapeutic targets for ALI/ARDS after cardiac surgery.

Pentraxin 3 in Circulating Microvesicles: a Potential Biomarker for Acute Heart Failure After Cardiac Surgery with Cardiopulmonary Bypass.

The aim of this study was to investigate whether pentraxin 3 (PTX3) in microvesicles (MVs) can be a valuable biomarker for the prediction of acute heart failure (AHF) after cardiac surgery with cardiopulmonary bypass (CPB). One hundred and twenty-four patients undergoing cardiac surgery with CPB were included and analyzed (29 with AHF and 95 without AHF). The concentrations of PTX3 in MVs isolated from plasma were measured by ELISA kits before, 12 h, and 3 days after surgery. Patients' demographics, medical history, surgical data, and laboratory results were collected. The levels of PTX3 in MVs were significantly elevated during perioperative surgery, which was increased more in the AHF group. The concentrations of PTX3 in MVs at postoperative 12 h were independent risk factors for AHF with the area under the ROC curve of 0.920. The concentration of PTX3 in MVs may be a novel biomarker for prediction of AHF after cardiac surgery.

Size Distribution of Microparticles: A New Parameter to Predict Acute Lung Injury After Cardiac Surgery With Cardiopulmonary Bypass.

Background: Acute lung injury (ALI) is a common complication after cardiac surgery with cardiopulmonary bypass (CPB). No precise way, however, is currently available to predict its occurrence. We and others have demonstrated that microparticles (MPs) can induce ALI and were increased in patients with ALI. However, whether MPs can be used to predict ALI after cardiac surgery with CPB remains unknown. Methods: In this prospective study, 103 patients undergoing cardiac surgery with CPB and 53 healthy subjects were enrolled. MPs were isolated from the plasma before, 12 h after, and 3 d after surgery. The size distributions of MPs were measured by the LitesizerTM 500 Particle Analyzer. The patients were divided into two subgroups (ALI and non-ALI) according to the diagnosis of ALI. Descriptive and correlational analyzes were conducted between the size distribution of MPs and clinical data. Results: Compared to the non-ALI group, the size at peak and interquartile range (IQR) of MPs in patients with ALI were smaller, but the peak intensity of MPs is higher. Multivariate logistic regression analysis indicated that the size at peak of MPs at postoperative 12 h was an independent risk factor for ALI. The area under the curve (AUC) of peak diameter at postoperative 12 h was 0.803. The best cutoff value of peak diameter to diagnose ALI was 223.05 nm with a sensitivity of 88.0% and a negative predictive value of 94.5%. The AUC of IQR at postoperative 12 h was 0.717. The best cutoff value of IQR to diagnose ALI was 132.65 nm with a sensitivity of 88.0% and a negative predictive value of 92.5%. Combining these two parameters, the sensitivity reached 92% and the negative predictive value was 96%. Conclusions: Our findings suggested that the size distribution of MPs could be a novel biomarker to predict and exclude ALI after cardiac surgery with CPB.

Circulating endothelial microparticles: a promising biomarker of acute kidney injury after cardiac surgery with cardiopulmonary bypass.

BACKGROUND: Current diagnostic strategies for acute kidney injury (AKI) after cardiac surgery with cardiopulmonary bypass (CPB) are nonspecific and limited. Previously, we demonstrated that circulating microparticles (MPs) in patients with valve heart disease (VHD) and congenital heart diseases (CHD) induce endothelial dysfunction and neutrophil chemotaxis, which may result in kidney injury. We also found that circulating MPs increase after cardiac surgery with CPB and are related to cardiac function. However, the relationship between circulating MPs and AKI after CPB is unknown. METHODS: Eighty-five patients undergoing cardiac surgery with CPB were enrolled. Patients were divided into AKI and non-AKI groups based on the serum creatinine levels at 12 h and 3 d post-CPB. Circulating MPs were isolated from plasma, and their levels including its subtypes were detected by flow cytometer. Independent risk factors for the CPB-associated AKI (CPB-AKI) were determined by multivariate logistic regression analysis. Receiver operating characteristic (ROC) analysis was used to measure the prognostic potential of CPB-AKI. RESULTS: The morbidity of AKI at 12 h and 3 d after cardiac surgery with CPB was 40% and 31.76%, respectively. The concentrations of total MPs and platelet-derived MPs (PMP) remained unchanged at 12 h and then increased at 3 d post-CPB, while that of endothelial-derived MPs (EMP) increased at both time points. In patients with AKI, PMP and EMP were elevated compared with the patients without AKI. However, no significant change was detected on monocyte-derived MPs (MMP) at 12 h and 3 d post-CPB. The logistic regression analysis showed that EMP was the independent risk factor for AKI both at 12 h and 3 d post-CPB. The area under ROC for the concentrations of EMP at 12 h and 3 d post-CPB was 0.86 and 0.91, with the specificity up to 0.88 and 0.91, respectively. CONCLUSIONS: Circulating EMP may serve as a potential biomarker of AKI after cardiac surgery with CPB.

Simvastatin inhibits POVPC-mediated induction of endothelial-to-mesenchymal cell transition.

Endothelial-to-mesenchymal transition (EndMT), the process by which an endothelial cell (EC) undergoes a series of molecular events that result in a mesenchymal cell phenotype, plays an important role in atherosclerosis. 1-Palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine (POVPC), derived from the oxidation of 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphatidylcholine, is a proinflammatory lipid found in atherosclerotic lesions. Whether POVPC promotes EndMT and how simvastatin influences POVPC-mediated EndMT remains unclear. Here, we treated human umbilical vein ECs with POVPC, simvastatin, or both, and determined their effect on EC viability, morphology, tube formation, proliferation, and generation of NO and superoxide anion (O2•-). Expression of specific endothelial and mesenchymal markers was detected by immunofluorescence and immunoblotting. POVPC did not affect EC viability but altered cellular morphology from cobblestone-like ECs to a spindle-like mesenchymal cell morphology. POVPC increased O2- generation and expression of alpha-smooth muscle actin, vimentin, Snail-1, Twist-1, transforming growth factor-beta (TGF-ß), TGF-ß receptor II, p-Smad2/3, and Smad2/3. POVPC also decreased NO production and expression of CD31 and endothelial NO synthase. Simvastatin inhibited POVPC-mediated effects on cellular morphology, production of O2•- and NO, and expression of specific endothelial and mesenchymal markers. These data demonstrate that POVPC induces EndMT by increasing oxidative stress, which stimulates TGF-ß/Smad signaling, leading to Snail-1 and Twist-1 activation. Simvastatin inhibited POVPC-induced EndMT by decreasing oxidative stress, suppressing TGF-ß/Smad signaling, and inactivating Snail-1 and Twist-1. Our findings reveal a novel mechanism of atherosclerosis that can be inhibited by simvastatin.

Circulating extracellular vesicles from patients with valvular heart disease induce neutrophil chemotaxis via FOXO3a and the inhibiting role of dexmedetomidine.

We previously demonstrated that circulating extracellular vesicles (EVs) from patients with valvular heart disease (VHD; vEVs) contain inflammatory components and inhibit endothelium-dependent vasodilation. Neutrophil chemotaxis plays a key role in renal dysfunction, and dexmedetomidine (DEX) can reduce renal dysfunction in cardiac surgery. However, the roles of vEVs in neutrophil chemotaxis and effects of DEX on vEVs are unknown. Here, we investigated the impact of vEVs on neutrophil chemotaxis in kidneys and the influence of DEX on vEVs. Circulating EVs were isolated from healthy subjects and patients with VHD. The effects of EVs on chemokine generation, forkhead box protein O3a (FOXO3a) pathway activation and neutrophil chemotaxis on cultured human umbilical vein endothelial cells (HUVECs) and kidneys in mice and the influence of DEX on EVs were detected. vEVs increased FOXO3a expression, decreased phosphorylation of Akt and FOXO3a, promoted FOXO3a nuclear translocation, and activated the FOXO3a signaling pathway in vitro. DEX pretreatment reduced vEV-induced CXCL4 and CCL5 expression and neutrophil chemotaxis in cultured HUVECs via the FOXO3a signaling pathway. vEVs were also found to suppress Akt phosphorylation and activate FOXO3a signaling to increase plasma levels of CXCL4 and CCL5 and neutrophil accumulation in kidney. The overall mechanism was inhibited in vivo with DEX pretreatment. Our data demonstrated that vEVs induced CXCL4-CCL5 to stimulate neutrophil infiltration in kidney, which can be inhibited by DEX via the FOXO3a signaling. Our findings reveal a unique mechanism involving vEVs in inducing neutrophils chemotaxis and may provide a novel basis for using DEX in reducing renal dysfunction in valvular heart surgery.

Microparticles (Exosomes) and Atherosclerosis.

PURPOSE OF REVIEW: This review summarizes the effects of microparticles and exosomes in the progression of atherosclerosis and the prospect for their diagnostic and therapeutic potentials. RECENT FINDINGS: Microparticles and exosomes can induce endothelial dysfunction, vascular inflammation, coagulation, thrombosis, and calcification via their components of proteins and noncoding RNAs, which may promote the progression of atherosclerosis. The applications of microparticles and exosomes become the spotlight of clinical diagnosis and therapy. Microparticles and exosomes are members of extracellular vesicles, which are generated in various cell types by different mechanisms of cell membrane budding and multivesicular body secretion, respectively. They are important physiologic pathways of cell-to-cell communication in vivo and act as messengers accelerating or alleviating the process of atherosclerosis. Microparticles and exosomes may become diagnostic biomarkers and therapeutic approaches of atherosclerosis.

Overexpression of inducible nitric oxide synthase in the diabetic heart compromises ischemic postconditioning.

Ischemia postconditioning (PTC) can reduce myocardial ischemia/reperfusion injury. However, the effectiveness of PTC cardioprotection is reduced or lost in diabetes and the mechanisms are largely unclear. Hyperglycemia can induce overexpression of inducible nitric oxide synthesis (iNOS) in the myocardium of diabetic subjects. However, it is unknown whether or not iNOS especially its overexpression plays an important role in the loss of cardioprotection of PTC in diabetes. C57BL6 and iNOS-/- mice were treated with streptozotocin to induce diabetes. Part of diabetic C57BL6 mice were also treated with an iNOS specific inhibitor, 1400 W. Mice were subjected to myocardial ischemia/ reperfusion with/without PTC. The hemodynamic parameters, plasma levels of cardiac troponin T (cTnT), TNF-α, IL-6 and nitric oxide (NO) were monitored. The myocardial infarct size, superoxide anion (O2-) generation, nitrotyrosine production and apoptosis were measured. The expression of phosphorylated Akt, endothelial NOS (eNOS), iNOS and Erk1/2 in ischemic heart were detected by immunoblot analysis. In diabetic C57BL6 and iNOS-/- mice, the post-ischemic hemodynamics were impaired, the cTnT, TNF-α, IL-6 level, myocardial infarct size, apoptotic index, O2- and nitrotyrosine generation were increased and the Akt/eNOS signal pathways were inhibited. PTC improved hemodynamic parameters, reduced cTnT level, myocardial infarct size, apoptotic index, O2- and nitrotyrosine generation and activated Akt/eNOS and Erk1/2 signal pathways in both non-diabetic C57BL6 and iNOS-/- mice as well as diabetic iNOS-/- mice, but not in diabetic C57BL6 mice. PTC also increased NO production in both non-diabetic and diabetic C57BL6 and iNOS-/- mice, and enhanced iNOS expression in non-diabetic C57BL6 mice. 1400 W restored the cardioprotection of PTC in diabetic C57BL6 mice. Our data demonstrated that PTC reduced myocardial ischemia/reperfusion injury in non-diabetic mice but not C57BL6 diabetic mice. Deletion of iNOS restored the cardioprotection of PTC in diabetic mice. Our findings suggest that iNOS plays a key role in the reduction of cardioprotection of PTC in diabetes and may provide a therapeutic target for diabetic patients.

Protein Compositions Changes of Circulating Microparticles in Patients With Valvular Heart Disease Subjected to Cardiac Surgery Contribute to Systemic Inflammatory Response and Disorder of Coagulation.

We recently demonstrated that circulating microparticles (MPs) from patients with valvular heart diseases (VHD) subjected to cardiac surgery impaired endothelial function and vasodilation. However, it is unknown whether or not the protein composition of these circulating MPs actually changes in response to the disease and the surgery. Circulating MPs were isolated from age-matched control subjects (n = 50) and patients (n = 50) with VHD before and 72 h after cardiac surgery. Proteomics study was performed by liquid chromatography and mass spectrometry combined with isobaric tags for relative and absolute quantification technique. The differential proteins were identified by ProteinPilot, some of which were validated by Western blotting. Bio-informatic analysis of differential proteins was carried out. A total of 849 proteins were identified and 453 proteins were found in all three groups. Meanwhile, 165, 39, and 80 proteins were unique in the control, pre-operation, and postoperation groups respectively. The unique proteins were different in localization, molecular function, and biological process. The pro-inflammatory proteins were increased in VHD patients and more so postoperatively. Proteins related to coagulation were dramatically changed before and after surgery. The protein composition of circulating MPs was changed in patients with VHD undergoing cardiac surgery, which may lead to activation of the systemic inflammatory response and disorders of coagulation.

The oxidized phospholipid POVPC impairs endothelial function and vasodilation via uncoupling endothelial nitric oxide synthase.

Endothelial dysfunction is an early stage of atherosclerosis. We recently have shown that 25-hydroxycholesterol found in atherosclerotic lesions could impair endothelial function and vasodilation by uncoupling and inhibiting endothelial nitric oxide synthase (eNOS). 1-Palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine (POVPC), the oxidation product of oxidized low-density lipoprotein, is another proinflammatory lipid and has also been found in atherosclerotic lesions. However, whether POVPC promotes atherosclerosis like 25-hydroxycholesterol remains unclear. The purpose of this study was to explore the effects of POVPC on endothelial function and vasodilation. Human umbilical vein endothelial cells (HUVECs) were incubated with POVPC. Endothelial cell proliferation, migration and tube formation were measured. Nitric oxide (NO) production and superoxide anion generation (O2-) were determined. The expression and phosphorylation of endothelial nitric oxide synthase (eNOS), AKT, PKC-ßII and P70S6K as well as the association of eNOS and heat shock protein 90 (HSP90) were detected by immunoblotting and immunoprecipitation. Endothelial cell apoptosis was monitored by TUNEL staining. The expression of Bcl-2, Bax, and Cleaved Caspase 3 were detected by immunoblotting. Finally, aortic ring from C57BL6 mice were isolated and treated with POVPC and the endothelium-dependent vasodilation was evaluated. POVPC significantly inhibited HUVECs proliferation, migration, tube formation, decreased NO production but increased O2- generation. POVPC inhibited the phosphorylation of Akt and eNOS at Ser1177, increased activation of PKC-ßII, P70S6K and the phosphorylation of eNOS at Thr495, reduced the association of HSP90 with eNOS. Meanwhile, POVPC induced endothelial cell apoptosis by inhibiting Bcl-2 expression, increasing Bax and cleaved caspase-3 expressions as well as caspase-3 activity and impaired endothelium-dependent vasodilation. These data demonstrated that POVPC impaired endothelial function by uncoupling and inhibiting eNOS as well as by inducing endothelial cell apoptosis. Therefore, POVPC may play an important role in the development of atherosclerosis and may be considered as a potential therapeutic target for atherosclerosis.

High density lipoprotein from coronary artery disease patients caused abnormal expression of long non-coding RNAs in vascular endothelial cells.

Increased evidence has showed that normal high density lipoprotein (HDL) could convert to dysfunctional HDL in diseases states including coronary artery disease (CAD), which regulated vascular endothelial cell function differently. Long non-coding RNAs (lncRNAs) play an extensive role in various important biological processes including endothelial cell function. However, whether lncRNAs are involved in the regulation of HDL metabolism and HDL-induced changes of vascular endothelial function remains unclear. Cultured human umbilical vein endothelial cells (HUVECs) were treated with HDL from healthy subjects and patients with CAD and hypercholesterolemia for 24 h, then the cells were collected for lncRNA-Seq and the expressions of lncRNAs, genes and mRNAs were identified. The bioinformatic analysis was used to evaluate the relationship among lncRNAs, encoding genes and miRNAs. HDL from healthy subjects and patients with CAD and hypercholesterolemia leaded to different expressions of lncRNAs, genes and mRNAs, and further analysis suggested that the differentially expressed lncRNAs played an important role in the regulation of vascular endothelial function. Thus, HDL from patients with CAD and hypercholesterolemia could cause abnormal expression of lncRNAs in vascular endothelial cells to affect vascular function.

Endothelial microparticles are increased in congenital heart diseases and contribute to endothelial dysfunction.

BACKGROUND: We previously demonstrated that endothelial microparticles (EMPs) are increased in mitral valve diseases and impair valvular endothelial cell function. Perioperative systemic inflammation is an important risk factor and complication of cardiac surgery. In this study, we investigate whether EMPs increase in congenital heart diseases to promote inflammation and endothelial dysfunction. METHODS: The level of plasma EMPs in 20 patients with atrial septal defect (ASD), 23 patients with ventricular septal defect (VSD), and 30 healthy subjects were analyzed by flow cytometry. EMPs generated from human umbilical vascular endothelial cells (HUVECs) were injected into C57BL6 mice, or cultured with HUVECs without or with siRNAs targeting P38 MAPK. The expression and/or phosphorylation of endothelial nitric oxide synthase (eNOS), P38 MAPK, and caveolin-1 in mouse heart and/or in cultured HUVECs were determined. We evaluated generation of nitric oxide (NO) in mouse hearts, and levels of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in cultured HUVECs and in mice. RESULTS: EMPs were significantly elevated in patients with ASD and VSD, especially in those with pulmonary hypertension when compared with controls. EMPs increased caveolin-1 expression and P38 MAPK phosphorylation and decreased eNOS phosphorylation and NO production in mouse hearts. EMPs stimulated P38 MAPK expression, TNF-α and IL-6 production, which were all inhibited by siRNAs targeting P38 MAPK in cultured HUVECs. CONCLUSIONS: EMPs were increased in adult patients with congenital heart diseases and may contribute to increased inflammation leading to endothelial dysfunction via P38 MAPK-dependent pathways. This novel data provides a potential therapeutic target to address important complications of surgery of congenial heart disease.