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Background and aims The apolipoprotein A-I mimetic peptide D-4F, among its anti-atherosclerotic effects, improves vasodilation through mechanisms not fully elucidated yet. Methods Low-density lipoprotein (LDL) receptor null (LDLr−/−) mice were fed Western diet with or without D-4F. We then measured atherosclerotic lesion formation, endothelial nitric oxide synthase (eNOS) phosphorylation and its association with heat shock protein 90 (HSP90), nitric oxide (NO) and superoxide anion (O2•-) production, and tetrahydrobiopterin (BH4) and GTP-cyclohydrolase 1 (GCH-1) concentration in the aorta. Human umbilical vein endothelial cells (HUVECs) and aortas were treated with oxidized LDL (oxLDL) with or without D-4F; subsequently, BH4 and GCH-1 concentration, NO and O2•- production, eNOS association with HSP90, and endothelium-dependent vasodilation were measured. Results Unexpectedly, eNOS phosphorylation, eNOS-HSP90 association, and O2•- production were increased, whereas BH4 and GCH-1 concentration and NO production were reduced in atherosclerosis. D-4F significantly inhibited atherosclerosis, eNOS phosphorylation, eNOS-HSP90 association, and O2•- generation but increased NO production and BH4 and GCH-1 concentration. OxLDL reduced NO production and BH4 and GCH-1 concentration but enhanced O2•- generation and eNOS association with HSP90, and impaired endothelium-dependent vasodilation. D-4F inhibited the overall effects of oxLDL. Conclusions Hypercholesterolemia enhanced uncoupled eNOS activity by decreasing GCH-1 concentration, thereby reducing BH4 levels. D-4F reduced uncoupled eNOS activity by increasing BH4 levels through GCH-1 expression and decreasing eNOS phosphorylation and eNOS-HSP90 association. Our findings elucidate a novel mechanism by which hypercholesterolemia induces atherosclerosis and D-4F inhibits it, providing a potential therapeutic approach.

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.

Acute heart failure (AHF) is a severe complication after cardiac surgery with cardiopulmonary bypass (CPB). Although some AHF biomarkers have been used in clinic, they have limitations when applied in the prediction and diagnosis of AHF after cardiac surgery with CPB, and there are still no effective and specific biomarkers. We and other researchers have shown that circulating microparticles (MPs) increased in a variety of cardiovascular diseases. However, whether the concentration of circulating MPs could be a new biomarker for AHF after cardiac surgery remains unknown. Here, 90 patients undergoing cardiac surgery with CPB and 45 healthy subjects were enrolled. Patients were assigned into AHF (n=14) or non-AHF (n=76) group according to the diagnosis criteria of AHF. The concentrations of circulating MPs were determined before, as well as 12 h and 3 days after operation with nanoparticle tracking analysis technique. MPs concentrations in patients before surgery were significantly higher than those of healthy subjects. Plasma levels of MPs were significantly elevated at 12 h after surgery in patients with AHF, but not in those without AHF, and the circulating MPs concentrations at 12 h after surgery were higher in AHF group compared with non-AHF group. Logistic regression analysis indicated that MPs concentration at postoperative 12 h was an independent risk factor for AHF. The area under receiver operating characteristic curve for MPs concentration at postoperative 12 h was 0.81 and the best cut-off value is 5.20×108 particles mL-1 with a sensitivity of 93% and a specificity of 10%. These data suggested that the concentration of circulating MPs might be a new biomarker for the occurrence of AHF after cardiac surgery with CPB.

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.

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.

This review summarizes the effects of microparticles and exosomes in the progression of atherosclerosis and the prospect for their diagnostic and therapeutic potentials. 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.

Simvastatin treatment is cardioprotective in patients undergoing noncoronary artery cardiac surgery. However, the mechanisms by which simvastatin treatment protects the myocardium under these conditions are not fully understood. Seventy patients undergoing noncoronary cardiac surgery, 35 from a simvastatin treatment group and 35 from a control treatment group, were enrolled in our clinical study. Simvastatin (20 mg/d) was administered preoperatively for 5-7 days. Myocardial tissue biopsies were taken before and after surgery. Apoptosis was detected by TUNEL staining. The expressions of Bcl-2 and Bak in myocardial tissue were detected by immunoblotting. The expressions of miRNA and Bcl-2 mRNA were detected by quantitative real-time polymerase chain reaction assays. Cardiomyocytes were isolated from rat and cultured cells. MiR-15a-5p mimic was transfected into cardiomyocytes, and the Bcl-2 was detected by immunoblotting. TUNEL staining showed significantly less myocardial apoptosis in the simvastatin treatment group when compared with the control treatment group. Protein expression of Bcl-2 was increased in the simvastatin treatment group before surgery, and Bak expression was increased in the control treatment group after surgery. Further comparisons showed that Bcl-2/Bak ratios were reduced in the control treatment group but were not significantly changed in the simvastatin treatment group after surgery. Furthermore, microarray assays revealed that miR-15a-5p was significantly decreased by simvastatin treatment. This was validated by quantitative real-time polymerase chain reaction analysis. MiR-15a-5p was predicted to target Bcl-2 mRNA at nucleotide positions 2529-2536. This was validated by luciferase binding assays. Coincident with the change in miR-15a-5p, the mRNA expression of Bcl-2 was increased in the simvastatin treatment group. MiR-15a-5p mimic significantly inhibited Bcl-2 expression in cardiomyocytes. Our findings strongly suggest that simvastatin treatment preoperatively protected the myocardium in patients undergoing noncoronary artery cardiac surgery, at least in part, by inhibiting apoptosis via suppressing miR-15a-5p expression, leading to increasing expression of Bcl-2 and decreasing expression of Bak.

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-βII 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-βII, 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.

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-to-mesenchymal transition (EndMT), the process by which an endothelial cell 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 pro-inflammatory lipid found in atherosclerotic lesions. Whether POVPC promotes EndMT, and how simvastatin influences POVPC-mediated EndMT remains unclear. Here, we treated human umbilical vein endothelial cells with POVPC, simvastatin, or both, and determined their effect on endothelial cell viability, morphology, tube formation, proliferation, and generation of nitric oxide (NO) and superoxide anion (O2●-). Expression of specific endothelial and mesenchymal markers was detected by immunofluorescence and immunoblotting. POVPC did not affect endothelial cell viability, but altered cellular morphology from cobblestone-like endothelial cells 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-β), TGF-β receptor II, p-Smad2/3, and Smad2/3. POVPC also decreased NO production, and expression of CD31 and endothelial nitric oxide 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-β/Smad signaling, leading to Snail-1 and Twist-1 activation. Simvastatin inhibited POVPC-induced EndMT by decreasing oxidative stress, suppressing TGF-β/Smad signaling, and inactivating Snail-1 and Twist-1. Our findings reveal a novel mechanism of atherosclerosis that can be inhibited by simvastatin.

Normal high-density lipoprotein (nHDL) in normal, healthy subjects is able to promote angiogenesis, but the mechanism remains incompletely understood. HDL from patients with coronary artery disease may undergo a variety of oxidative modifications, rendering it dysfunctional; whether the angiogenic effect is mitigated by such dysfunctional HDL (dHDL) is unknown. We hypothesized that dHDL compromises angiogenesis. The angiogenic effects of nHDL and dHDL were assessed using endothelial cell culture, endothelial sprouts from cardiac tissue from C57BL/6 mice, zebrafish model for vascular growth and a model of impaired vascular growth in hypercholesterolemic low-density lipoprotein receptor null(LDLr-/-)mice. MiRNA microarray and proteomic analyses were used to determine the mechanisms. Lipid hydroperoxides were greater in dHDL than in nHDL. While nHDL stimulated angiogenesis, dHDL attenuated these responses. Protein and miRNA profiles in endothelial cells differed between nHDL and dHDL treatments. Moreover, nHDL suppressed miR-24-3p expression to increase vinculin expression resulting in nitric oxide (NO) production, whereas dHDL delivered miR-24-3p to inhibit vinculin expression leading to superoxide anion (O2•-) generation via scavenger receptor class B type 1. Vinculin was required for endothelial nitric oxide synthase (eNOS) expression and activation and modulated the PI3K/AKT/eNOS and ERK1/2 signaling pathways to regulate nHDL- and VEGF-induced angiogenesis. Vinculin overexpression or miR-24-3p inhibition reversed dHDL-impaired angiogenesis. The expressions of vinculin and eNOS and angiogenesis were decreased, but the expression of miR-24-3p and lipid hydroperoxides in HDL were increased in the ischemic lower limbs of hypercholesterolemic LDLr-/- mice. Overexpression of vinculin or miR-24-3p antagomir restored the impaired-angiogenesis in ischemic hypercholesterolemic LDLr-/- mice. Collectively, nHDL stimulated vinculin and eNOS expression to increase NO production by suppressing miR-24-3p to induce angiogenesis, whereas dHDL inhibited vinculin and eNOS expression to enhance O2•- generation by delivering miR-24-3p to impair angiogenesis, and that vinculin and miR-24-3p may be therapeutic targets for dHDL-impaired angiogenesis., Graphical abstract Image 1, Highlights • nHDL and dHDL regulated angiogenesis differently via alterations in vinculin expression. • nHDL suppressed miR-24-3p to increase vinculin expression to stimulate NO production. • dHDL delivered miR-24-3p to inhibit vinculin expression to enhance O2.•- generation. • Vinculin and miR-24-3p may be therapeutic targets for dHDL-impaired angiogenesis. • Cell-free assay may be used to measure the oxidative levels of HDL.

Cold cardioplegia is used to induce heart arrest during cardiac surgery. However, endothelial function may be compromised after this procedure. Accordingly, interventions such as adenosine, that mimic the effects of preconditioning, may minimize endothelial injury. Herein, we investigated whether adenosine prevents cold-induced injury to the endothelium. Cultured human cardiac microvascular endothelial cells were treated with adenosine for different durations. Phosphorylation and expression of endothelial nitric oxide synthase (eNOS), p38MAPK, ERK1/2, and p70S6K6 were measured along with nitric oxide (NO) production using diaminofluorescein-2 diacetate (DAF-2DA) probe. Cold-induced injury by hypothermia to 4°C for 45 minutes to mimic conditions of cold cardioplegia during open heart surgery was induced in human cardiac microvascular endothelial cells. Under basal conditions, adenosine stimulated NO production, eNOS phosphorylation at serine 1177 from 5 minutes to 4 hours and inhibited eNOS phosphorylation at threonine 495 from 5 minutes to 6 hours, but increased phosphorylation of ERK1/2, p38MAPK, and p70S6K only after exposure for 5 minutes. Cold-induced injury inhibited NO production and the phosphorylation of the different enzymes. Importantly, adenosine prevented these effects of hypothermic injury. Our data demonstrated that adenosine prevents hypothermic injury to the endothelium by activating ERK1/2, eNOS, p70S6K, and p38MAPK signaling pathways at early time points. These findings also indicated that 5 minutes after administration of adenosine or release of adenosine is an important time window for cardioprotection during cardiac surgery.

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. Electronic supplementary material The online version of this article (doi:10.1186/s12967-016-1087-2) contains supplementary material, which is available to authorized users.

Normal high density lipoprotein (HDL) protects vascular function; however these protective effects of HDL may absent in valvular heart disease (VHD). Because vascular function plays an important role in maintaining the circulation post-cardiac surgery and some patients are difficult to stabilize, we hypothesized that a deleterious vascular effect of HDL may contribute to vascular dysfunction in VHD patients following surgery. HDL was isolated from age-match 28 healthy subjects and 84 patients with VHD and during cardiac surgery. HDL pro-inflammation index was measured and the effects of HDL on vasodilation, protein interaction, generation of nitric oxide (NO) and superoxide were determined. Patients with VHD received either simvastatin (20mg/d) or routine medications, and endothelial effects of HDL were characterized. HDL inflammation index significantly increased in VHD patients and post-cardiac surgery. HDL from VHD patients and post-cardiac surgery significantly impaired endothelium-dependent vasodilation, inhibited both Akt and endothelial nitric oxide synthase (eNOS) phosphorylation at S1177, eNOS associated with heat shock protein 90 (HSP90), NO production and increased eNOS phosphorylation at T495 and superoxide generation. Simvastatin therapy partially reduced HDL inflammation index, improved the capacity of HDL to stimulate eNOS and Akt phosphorylation at S1177, eNOS associated with HSP90, NO production, reduced eNOS phosphorylation at T495 and superoxide generation, and improved endothelium-dependent vasodilation. Our data demonstrated that HDL from VHD patients and cardiac surgery contributed to endothelial dysfunction through uncoupling of eNOS. This deleterious effect can be reversed by simvastatin, which improves the vasoprotective effects of HDL. Targeting HDL may be a therapeutic strategy for maintaining vascular function and improving the outcomes post-cardiac surgery.

Mitral valve endothelial cells are important for maintaining lifelong mitral valve integrity and function. Plasma endothelial microparticles (EMPs) increased in various pathological conditions related to activation of endothelial cells. However, whether EMPs will increase in mitral valve disease and their relationship remains unclear. Here, 81 patients with mitral valve disease and 45 healthy subjects were analyzed for the generation of EMPs by flow cytometry. Human mitral valve endothelial cells (HMVECs) were treated with EMPs. The phosphorylation of Akt and endothelial nitric oxide synthase (eNOS), the association of eNOS and heat shock protein 90 (HSP90), and the generation of nitric oxide (NO) and superoxide anion (O2˙−) were measured. EMPs were increased significantly in patients with mitral valve disease compared with those in healthy subjects. EMPs were negatively correlated with mitral valve area in patients with isolated mitral stenosis. EMPs were significantly higher in the group with severe mitral regurgitation than those in the group with mild and moderate mitral regurgitation. Furthermore, EMPs were decreased dramatically in both Akt and eNOS phosphorylation and the association of HSP90 with eNOS in HMVECs. EMPs decreased NO production but increased O2˙− generation in HMVECs. Our data demonstrated that EMPs were significantly increased in patients with mitral valve disease. The increase of EMPs can in turn impair HMVEC function by inhibiting the Akt/eNOS-HSP90 signaling pathway. These findings suggest that EMPs may be a therapeutic target for mitral valve disease.

Endothelial dysfunction is a key early step in atherosclerosis. 25-Hydroxycholesterol (25-OHC) is found in atherosclerotic lesions. However, whether 25-OHC promotes atherosclerosis is unclear. Here, we hypothesized that 25-OHC, a proinflammatory lipid, can impair endothelial function, which may play an important role in atherosclerosis. Bovine aortic endothelial cells were incubated with 25-OHC. Endothelial cell proliferation, migration, and tube formation were measured. Nitric oxide (NO) production and superoxide anion generation were determined. The expression and phosphorylation of endothelial NO synthase (eNOS) and Akt as well as the association of eNOS and heat shock protein (HSP)90 were detected by immunoblot analysis and immunoprecipitation. Endothelial cell apoptosis was monitored by TUNEL staining and caspase-3 activity, and expression of Bcl-2, Bax, cleaved caspase-9, and cleaved caspase-3 were detected by immunoblot analysis. Finally, aortic rings from Sprague-Dawley rats were isolated and treated with 25-OHC, and endothelium-dependent vasodilation was evaluated. 25-OHC significantly inhibited endothelial cell proliferation, migration, and tube formation. 25-OHC markedly decreased NO production and increased superoxide anion generation. 25-OHC reduced the phosphorylation of Akt and eNOS and the association of eNOS and HSP90. 25-OHC also enhanced endothelial cell apoptosis by decreasing Bcl-2 expression and increasing cleaved caspase-9 and cleaved caspase-3 expressions as well as caspase-3 activity. 25-OHC impaired endothelium-dependent vasodilation. These data demonstrated that 25-OHC could impair endothelial function by uncoupling and inhibiting eNOS activity as well as by inducing endothelial cell apoptosis. Our findings indicate that 25-OHC may play an important role in regulating atherosclerosis.

l-Arginine can attenuate pulmonary hypertension (PH) by a mechanism that are not fully understood. This study investigated the molecule mechanism of l-arginine attenuating PH. Sprague Dawley rats were treated with monocrotaline (MCT) with or without l-arginine for 3 or 5 wk. Right ventricular systolic pressure (RVSP), right heart hypertrophy, survival rate, pulmonary artery wall thickness, nitric oxide (NO) concentration, and superoxide anion (O2·−) generation in the lung were measured. Expressions of endothelial nitric oxide synthase (eNOS) and heat shock protein 90 (HSP90), phosphorylation of eNOS at Ser1177, and the association of eNOS and HSP90 in the lung were determined by Western blot and immunoprecipitation experiments. MCT increased RVSP, right heart hypertrophy, mortality, pulmonary artery wall thickness, and O2·− generation and decreased eNOS and HSP90 expression and association, phosphorylation of eNOS at Ser1177, and NO production. l-Arginine decreased RVSP, right heart hypertrophy, mortality, O2·− generation, and pulmonary artery wall thickness and increased NO production. l-Arginine increased eNOS expression, phosphorylation of eNOS at Ser1177, and association of eNOS and HSP90 without significantly altering HSP90 expression. l-Arginine may act through three pathways, providing a substrate for NO generation, preserving eNOS expression/phosphorylation, and maintaining the association of eNOS and HSP90, which allows restoration of eNOS activity and coupling activity, to maintain the balance between NO and O2·− and delay the development of PH.

Objective: Circulating microparticles (MPs) from some diseases play important roles on inflammation, coagulation and vascular injury. The vascular function is quite important for maintaining the circulation after cardiac surgery. However, the impact of MPs generated from valvular heart disease (VHD) and cardiac surgery on vascular function remains unknown. The present study is to investigate the impact of such MPs on vasodilation. Methods: MPs were isolated from age-match healthy subjects and patients with VHD before and at 12 h, 72 h after cardiac surgery. Effects of MPs on vasodilation of mice aorta, the phosphorylation and expression of Akt, protein kinase C βII (PKC-βII), p70S6K, endothelial nitric oxide synthase (eNOS) and caveolin-1, the association of eNOS with heat shock protein 90 (HSP90), generation of nitric oxide (NO) and superoxide anion (O2•–) of human umbilical vein endothelial cells (HUVECs) were determined. Results: Compared with MPs from healthy subjects, MPs from VHD patients before cardiac surgery can impair endothelium-dependent vasodilation, inhibit phosphorylation of Akt and eNOS, enhance expression of caveolin-1, and activation of PKC-βII, p70S6K, and reduced association of HSP90 with eNOS, decrease NO production and increase O2•– generation. Those effects were even stronger when treated with MPs from post- cardiac surgery. Conclusion: Our data demonstrated that circulating MPs generated before and after cardiac surgery in VHD patients contributed to endothelial dysfunction by inhibiting and uncoupling of eNOS activity. Circulating MPs may be a potential therapeutic target for maintenance of vascular function post-cardiac surgery.

Objective Vascular function is very important for maintaining circulation after cardiac surgery. Circulating microparticles (MPs) generated in various diseases play important roles in causing inflammation, coagulation, and vascular injury. However, the impact of MPs generated from patients who have valvular heart disease (VHD), before and after cardiac surgery, on vascular function remains unknown. This study is designed to investigate the impact of such MPs on vasodilation. Methods Microparticles were isolated from age-matched healthy subjects and patients who had VHD, before cardiac surgery, and at 12 hours and 72 hours afterward. The number of MPs was measured and compared. Effects evaluated were of the impact of MPs on: vasodilation of mice aorta; the phosphorylation and expression of Akt, endothelial nitric oxide synthase (eNOS), protein kinase C-βII (PKC-βII), and p70 ribosomal protein S6 kinase (p70S6K); expression of caveolin-1; the association of eNOS with heat shock protein 90 (HSP90); and generation of nitric oxide and superoxide anion of human umbilical vein endothelial cells. Results Compared with the healthy subjects, VHD patients had significantly higher levels of circulating MPs and those MPs before cardiac surgery can: impair endothelium-dependent vasodilation; inhibit phosphorylation of Akt and eNOS; increase activation of PKC-βII and p70S6K; enhance expression of caveolin-1; reduce the association of HSP90 with eNOS; decrease nitric oxide production, and increase superoxide anion generation. These deleterious effects were even stronger in postoperative MPs. Conclusions Our data demonstrate that MPs generated from VHD patients before and after cardiac surgery contributed to endothelial dysfunction, by uncoupling and inhibiting eNOS. Circulating MPs are potential therapeutic targets for the maintenance of vascular function postoperatively.

Vitamin E (VitE) only prevented cardiovascular diseases in some patients and the mechanisms remain unknown. VitE levels can be affected by aging and gender. We hypothesize that age and gender can influence VitE’s cardioprotective effect. Mice were divided into 4 groups according to age and gender, and each group of mice were divided into a control group and a VitE group. The mice were administered water or VitE for 21 days; Afterward, the cardiac function and myocardial infarct size and cardiomyocyte apoptosis were measured after myocardial ischemia reperfusion(MI/R). VitE may significantly improved cardiac function in young male mice and aged female mice by enhancing ERK1/2 activity and reducing JNK activity. Enhanced expression of HSP90 and Bcl-2 were also seen in young male mice. No changes in cardiac function and cardiac proteins were detected in aged male mice and VitE was even liked to exert a reverse effect in cardiac function in young mice by enhancing JNK activity and reducing Bcl-2 expression. Those effects were in accordance with the changes of myocardial infarction size and cardiomyocyte apoptosis in each group of mice. VitE may reduce MI/R injury by inhibiting cardiomyocyte apoptosis in young male mice and aged female mice but not in aged male mice. VitE was possibly harmful for young female mice, shown as increased cardiomyocyte apoptosis after MI/R. Thus, we speculated that the efficacy of VitE in cardiac protection was associated with age and gender.

Aims: Previous clinical trials designed to stimulate coronary vascular growth (therapeutic angiogenesis) using growth factors, e.g, vascular endothelial growth factor (VEGF), in patients with coronary artery disease (CAD) were unsuccessful. Although many reasons were postulated for the failure, e.g., endothelial dysfunction, refractory response to VEGF, no consensus was achieved to explain these unsuccessful attempts. To further interrogate this conundrum, we found that high density lipoprotein from healthy subjects (nHDL) is pro-angiogenic; however, HDL from patients with CAD (pHDL) has oxidative modifications. Accordingly we postulate that this modified pHDL is proinflammatory and “switches” to an anti-angiogenic effect. To test this possibility we determined the angiogenic capacity of pHDL and of pHDL on VEGF induced-angiogenesis. Methods and Results: Human umbilical vein endothelial cells (HUVECs) were cultured; murine hearts were isolated and sectioned, and the sections were cultured under condition in which endothelial cells sprout from the sections. HUVECs and the heart sections were treated with nHDL (100 μg/ml) or pHDL (100 μg/ml) with or without VEGF stimulation. We measured proliferation, migration, tube formation of HUVECs, and number and length of sprouts from the sections. pHDL (compared to nHDL) reduced endothelial cell proliferation, migration, tube formation, and the length of the sprouts from the sections under basal conditions and also in response to VEGF (all P− ) generation, phosphorylation of endothelial nitric oxide synthase (eNOS), and Akt in HUVECs. Compared to nHDL, pHDL reduced the phosphorylation of eNOS, and Akt; and, also inhibited NO production, but increased eNOS-dependent O2[[Unable to Display Character: ˙]] − generation. Conclusions: HDL from patients with CAD impaired VEGF-induced angiogenesis via eNOS/Akt signaling pathway. Taken together, our findings suggest that oxidatively modified HDL from patients with CAD may be a contributing factor to reasons why therapeutic angiogenesis trials failed using VEGF recombinant protein or VEGF gene therapy.

Background: The vascular effects of high density lipoprotein (HDL) may be highly heterogeneous. Normal HDL protects vascular function. Vascular function plays an important role in maintaining the circulation stable post-cardiac surgery. The vascular effects of HDL in valvular heart disease (VHD) and cardiac surgery remain unknown. The present study is to determine the vascular effects of HDL from VHD and cardiac surgery. Methods: HDL was isolated from healthy subjects and patients with VHD and during cardiac surgery. HDL inflammation index was measured by cell free assay. Effects of HDL on vasodilation, protein interaction, generation of nitric oxide (NO) and superoxide anion (O 2 ·– ) were determined. Results: HDL inflammation index significantly increased in VHD patients and post-cardiac surgery. HDL from VHD patients and post-cardiac surgery significantly impaired endothelium-dependent vasodilation, inhibited both Akt and endothelial nitric oxide synthase (eNOS) phosphorylation, eNOS associated with heat shock protein 90, NO production and increased O 2 ·– generation. Conclusions: The vasoprotective effects of HDL from VHD patients and cardiac surgery is impaired by uncoupled eNOS activity. HDL may be a therapeutic target for maintaining the stable circulation in post-cardiac surgery.

Background Deficiency in number and activity of circulating EPCs is associated with reduced arterial elasticity in humans with advancing aging. Physical exercise can increase the number and activity of circulating EPCs in humans. Here we investigated whether regular exercise-induced enhanced circulating endothelial progenitor cells (EPCs) improves age-related decline in arterial elasticity in healthy men. Methods In a cross-sectional study, the number and activity of circulating EPCs as well as brachial-ankle pulse wave velocity (baPWV) of young and older sedentary or endurance-trained healthy men were studied. Then we observed the effect of regular exercise on circulating EPCs and baPWV of 10 older and 10 young sedentary healthy men. Results In both sedentary and endurance-trained men, the number and activity of circulating EPCs were significantly low in older men compared with young men, which was paralleled to increased baPWV. After three months of regular exercise, the number and activity of circulating EPCs increased, and the baPWV of 10 older and 10 young sedentary healthy men decreased. However, the increased number and activity of circulating EPCs and decreased baPWV of older sedentary healthy men were higher. There was a close correlation between circulating EPCs and baPWV. Multivariate analysis identified proliferative activity of circulating EPCs as an independent predictor of baPWV. Conclusions The present study demonstrates for the first time that regular physical exercise-induced enhanced circulating EPCs attenuates age-related decline in arterial elasticity in healthy men. These findings provide novel insights into the protective effects of exercise on age-related vascular injury.

Therapeutic angiogenesis remains unsuccessful in coronary artery disease. It is known that plasma endothelium-derived microparticles (EMPs) are increased in coronary artery disease and that hypercholesterolemia can inhibit angiogenesis. We evaluated the relationship between EMPs and hypercholesterolemia in the impairment of angiogenesis. EMPs isolated from human umbilical vein endothelial cells were injected into low-density lipoprotein receptor-null (LDLr−/−) mice fed a Western diet for 2 wk and C57BL6 mice for 6 h or were directly added to the tissue culture media. Hearts isolated from mice were sectioned and cultured, and endothelial tube formation was measured. The expression and phosphorylation of endothelial NO synthase (eNOS) and the generation of NO in the hearts were determined. Angiogenesis was inhibited by pathophysiological concentrations of EMPs but not physiological concentrations of EMPs in hearts from C57BL6 mice. However, angiogenesis was inhibited by EMPs at both physiological and pathophysiological concentrations of EMPs in hearts from hypercholesterolemic LDLr−/−mice. Pathophysiological concentrations of EMPs decreased eNOS phosphorylation at Ser1177and NO generation without altering eNOS expression in hearts from C57BL6 mice. Both physiological and pathophysiological concentrations of EMPs decreased not only eNOS phosphorylation at Ser1177and NO generation, but eNOS expression in hypercholesterolemic hearts from LDLr−/−mice. These data demonstrated that pathophysiological concentrations of EMPs could inhibit angiogenesis in hearts by decreasing eNOS activity. EMPs and hypercholesterolemia mutually enhanced their inhibitory effect of angiogenesis by inducing eNOS dysfunction. Our findings suggest a novel mechanism by which hypercholesterolemia impairs angiogenesis.

To investigate the importance of the specific structure of L-4F, an apolipoprotein A-1 mimetic, in inhibiting atherosclerosis. The study was designed to compare the effect of L-4F and scramble-4F (SC-4F) in preventing low density lipoprotein (LDL) induced endothelial cell dysfunction. L-4F and SC-4F has the same amino acids but different nucleotide sequence.Bovine aortic endothelial cells (BAEC) were incubated with single L-4F 10 microg/ml or SC-4F 10 microg/ml, or LDL 6.2 mmol/L in the absence or presence of L-4F 10 microg/ml or SC-4F 10 microg/ml for 24 h and assayed for (1) changes of superoxide anion (O2-*) generation in BAEC by superoxide dismutase (SOD)-inhibitable ferricytochrome C reduction, and (2) the production of nitric oxide (NO) in BAEC by ozone chemiluminescence with VCL3.(1) L-4F and SC-4F themselves had no affect on BAEC O2-* generation. (2) LDL significantly increased O2-* generation in BAEC and LDL-induced O2-* generation was inhibited by pretreatment of LDL with L-4F. However, pretreatment of LDL with SC-4F had no effect on inhibition of LDL-induced O2-* generation. (3) LDL significantly inhibited NO generation in BAEC and pretreatment of LDL with L-4F could inhibit LDL-induced decrease of NO generation, but pretreatment of LDL with SC-4F still inhibited NO generation in BAEC.L-4F can prevent LDL induced endothelial cell dysfunction by maintaining the balance of NO and O2-*, but SC-4F doesn't. It suggests that the specific structure of L-4F may play a crucial role in preventing atherosclerosis and it may provide a new clue for searching a novel approach on prevention and therapeutics of atherosclerosis in the future.