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.

Spontaneous multivessel coronary artery spasm diagnosed with intravascular ultrasound imaging: A case report.

BACKGROUND: Coronary artery spasm is a major cause of myocardial ischemia. Although coronary artery spasm has been known for a long time, its mechanism has not yet been identified. Many clinicians, especially young clinicians pay less attention to coronary artery spasm, which may lead to some patients not being appropriately diagnosed and treated in time. We report a patient with spontaneous multivessel coronary artery spasm for more than 30 years diagnosed with intravascular ultrasound (IVUS) imaging. CASE SUMMARY: A 66-year-old Chinese male patient had chest squeezing at rest for more than 30 years. He had a history of cigarette smoking for more than 40 years and hypertension for 10 years. Before presenting at our institution, the patient had undergone coronary angiography 4 times and percutaneous transluminal coronary angioplasty procedures twice at other hospitals without a diagnosis of coronary artery spasm. However, his chest symptoms worsened. Spontaneous multivessel coronary artery spasm occurred during IVUS without provocation testing, and the IVUS image was recorded. Thus, the diagnosis of multifocal spontaneous coronary artery spasm was confirmed. The patient was placed on oral diltiazem, isosorbide mononitrate, and nicorandil to suppress coronary artery spasms. All medications were given at the maximum dosages tolerated by the patient. He was discharged after 5 d without complications. During the six-month follow-up period, the patient was symptom-free. CONCLUSION: Coronary artery spasm is still prevalent in Eastern countries. It is essential for clinicians to be aware of coronary artery spasm, which may be hard to detect and can be lethal, in order to diagnose and treat patients appropriately.

Microparticles from Patients with the Acute Coronary Syndrome Impair Vasodilatation by Inhibiting the Akt/eNOS-Hsp90 Signaling Pathway.

OBJECTIVES: Endothelial dysfunction is involved in the development of the acute coronary syndrome (ACS). Plasma microparticles(MPs) from other diseases have been demonstrated to initiate coagulation and endothelial dysfunction.However, whether MPs from ACS patients impair vasodilatation and endothelial function remains unclear. METHODS: Patients(n = 62) with ACS and healthy controls (n = 30) were recruited for MP isolation. Rat thoracic aortas were incubated with MPs from ACS patients or healthy controls to determine the effects of MPs on endothelial-dependent vasodilatation,the phosphorylation of Akt and endothelial nitric oxide synthase (eNOS), the interaction of eNOS with heat shock protein 90 (Hsp90), and nitric oxide (NO) and superoxide anion(O 2 ­ ) production. The origin of MPs was assessed by flow cytometry. RESULTS: MP concentrations were increased in patients with ACS compared with healthy controls. They were positively correlated with the degree of coronary artery stenosis. MPs from ACS patients impair endothelial-dependent vasodilatation, decrease both Akt and eNOS phosphorylation,decrease the interaction between eNOS and Hsp90,and decrease NO production but increase O 2 ­ generation in rat thoracic aortas. Endothelial-derived MPs and platelet-derived MPs made up nearly 75% of MPs. CONCLUSIONS: Our data indicate that MPs from ACS patients negatively affect endothelial-dependent vasodilatation via Akt/eNOS-Hsp90 pathways.

Circulating microparticles from patients with valvular heart disease and cardiac surgery inhibit endothelium-dependent vasodilation.

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-ßII (PKC-ßII), 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-ßII 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.

High density lipoprotein from patients with valvular heart disease uncouples endothelial nitric oxide synthase.

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.

A simple modification results in greater success in the model of coronary artery ligation and myocardial ischemia in mice.

Mouse models of myocardial ischemic preconditioning (IPC) and ischemic postconditioning (IPD) have proven to be very useful models of cardiovascular diseases. In 2010, Gao described a novel procedure without the aid of mechanical ventilation. However, the technique of heart externalization could not be applied to mouse models of IPC or IPD due to the limited time frame of the technique. We proposed a modified simple and safe method using lung recruitment and short-term ventilation to perform the procedure in mice with IPC or IPD. The mice were randomly divided into 4 groups: the modified groups, M-IPC and M-IPD, and the conventional groups, C-IPC and C-IPD. In the 2 modified groups, the mice were removed from the ventilator and allowed to resume breathing spontaneously upon completion of the lung recruitment and the rapid closure of the thorax. Our study demonstrated that the postoperative recovery time was significantly reduced for the modified groups compared with the 2 conventional groups. Moreover, the inflammatory damages were attenuated by the modified method compared with the conventional method. In addition, the modified method significantly increased the survival rates of mice with IPC or IPD. The modified method improved the survival rates of mouse models of myocardial ischemia.

Endothelial microparticles increase in mitral valve disease and impair mitral valve endothelial function.

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 (O(2)(∙-)) 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 O(2)(∙-) 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.

Simvastatin reduces myocardial injury undergoing noncoronary artery cardiac surgery: a randomized controlled trial.

OBJECTIVE: Myocardial injury during cardiac surgery is a major cause of perioperative morbidity and mortality. We determined whether perioperative statin therapy is cardioprotective in patients undergoing noncoronary artery cardiac surgery and the potential mechanisms. METHODS AND RESULTS: One hundred fifty-one patients undergoing noncoronary artery cardiac surgery were randomly assigned to either a statin group (n=77) or a control group (n=74). Simvastatin (20 mg) was administered preoperatively and postoperatively. Plasma were analyzed for troponin T, isoenzyme of creatine kinase, C-reaction protein, interleukin-6, interleukin-8, creatinine, and blood urea nitrogen. Cardiac echocardiography was performed. Endothelial nitric oxide synthase (eNOS), Akt, p38, heat shock protein 90, caveolin-1, and nitric oxide (NO) in the heart were detected. Simvastatin significantly reduced plasma troponin T, isoenzyme of creatine kinase, C-reaction protein, blood urea nitrogen , creatinine, interleukin-6, interleukin-8, and the requirement of inotropic postoperatively. Simvastatin increased NO production, the expression of eNOS and phosphorylation at serine1177, phosphorylation of Akt, expression of heat shock protein 90, heat shock protein 90 association with eNOS and decreased eNOS phosphorylation at threonine 495, phosphorylation of p38, and expression of caveolin-1. Simvastatin also improved cardiac function postoperatively. CONCLUSIONS: Perioperative statin therapy can improve cardiac function and renal function by reducing myocardial injury and inflammatory response through activating Akt-eNOS and attenuating p38 signaling pathways in patients undergoing noncoronary artery cardiac surgery. Clinical Trial Registration- URL: http://www.clinicaltrials.gov. Unique identifier: NCT01178710.

Endothelial nitric oxide synthase enhancer for protection of endothelial function from asymmetric dimethylarginine-induced injury in human internal thoracic artery.

OBJECTIVES: Endogenous nitric oxide synthase inhibitor asymmetric dimethylarginine is a cardiovascular risk factor that is elevated in patients with coronary artery disease. We hypothesized that novel endothelial nitric oxide synthase enhancer AVE3085 might improve the endothelial function altered by asymmetric dimethylarginine in the human internal thoracic artery. METHODS: Cumulative concentration-relaxation curves to acetylcholine (-11 to -5 log mol/L) were established in left internal thoracic artery rings (n = 65) from 27 patients undergoing coronary artery bypass grafting in precontraction induced by U46619 (-8 log mol/L) in the absence or presence of asymmetric dimethylarginine (100 µmol/L) or AVE3085 (30 µmol/L). Protein expressions of endothelial nitric oxide synthase and levels of superoxide anion production were detected. RESULTS: Maximal relaxation induced by acetylcholine was significantly attenuated by asymmetric dimethylarginine (12.7% ± 2.3% vs 35.3% ± 5.0% in control; P < .05) and significantly restored by AVE3085 (23.4% ± 2.8%; P < .05). AVE3085 also markedly restored endothelial nitric oxide synthase expression (0.29 ± 0.008; P = .012) reduced by asymmetric dimethylarginine (0.05 ± 0.04 vs 0.36 ± 0.03 in control; P = .014). Increased superoxide anion production by asymmetric dimethylarginine (2.97 ± 0.25 vs 0.51 ± 0.10 relative light units/[s/mg] in control; P < .05) was inhibited by AVE3805 (0.62 ± 0.104 relative light units/[s/mg]; P < .05). CONCLUSIONS: AVE3085 may restore endothelium-dependent relaxation reduced by asymmetric dimethylarginine through upregulation of endothelial nitric oxide synthase expression and inhibition of production of superoxide anion in human internal thoracic artery. These findings provide new insights into endothelial protection of coronary bypass grafting vessels to improve long-term patency of grafts.

dl-3n-Butylphthalide promotes angiogenesis via the extracellular signal-regulated kinase 1/2 and phosphatidylinositol 3-kinase/Akt-endothelial nitric oxide synthase signaling pathways.

We have previously demonstrated that dl-3n-butylphthalide (NBP) has a potential angiogenic activity. In this study, we investigated the angiogenic effect of NBP and the molecular mechanisms underlying NBP-mediated angiogenesis. Zebrafish embryos and human umbilical vein endothelial cells were treated with various doses of NBP and several signaling pathway inhibitors. NBP induced ectopic subintestinal vessel production in zebrafish embryos and induced invasion, migration, and endothelial cell tube formation of human umbilical vein endothelial cells in a dose-dependent manner. These NBP-induced angiogenic effects were partially suppressed by SU5402, a fibroblast growth factor receptor 1 inhibitor; U0126, an extracellular signal-regulated kinase 1/2 (ERK1/2) inhibitor; LY294002, a phosphatidylinositol 3-kinase inhibitor; 1L6-hydroxymethyl-chiro-inositol-2-(R)-2-O-methyl-3-O-octadecyl-sn-glycerocarbonate, an Akt inhibitor; cavtratin, an endothelial nitric oxide synthase (eNOS) inhibitor and completely inhibited by a combination of U0126 and LY294002. NBP enhanced phosphorylation of ERK1/2 and fibroblast growth factor receptor 2 expression, which were inhibited by U0126. NBP increased the phosphorylation of Akt and eNOS at serine 1177, which was blocked by LY294002. NBP-stimulated nitric oxide production, which was reduced by LY294002. Our data demonstrated that (1) NBP promoted angiogenesis and (2) the angiogenic effects of NBP were mediated by the ERK1/2 and phosphatidylinositol 3-kinase/Akt-eNOS signaling pathways. Our findings suggest that NBP could be a novel agent for therapeutic angiogenesis in ischemic diseases.

Remote ischaemic preconditioning reduces myocardial injury in patients undergoing heart valve surgery: randomised controlled trial.

OBJECTIVE: To determine whether remote ischaemic preconditioning (RIPC) is cardioprotective in patients undergoing heart valve replacement. DESIGN: Single-blinded, randomised controlled trial. SETTING: Tertiary referral hospital in China. PATIENTS: Adult patients (31-72 years) undergoing mitral valve, aortic valve or tricuspid valve surgery. INTERVENTIONS: Patients were randomised to either the RIPC (n=38) or control (n=35) group. After induction of anaesthesia, patients in the RIPC group underwent three 5 min cycles of right upper limb ischaemia, induced by an automated cuff-inflator placed on the upper arm and inflated to 200 mm Hg. Each cycle was interrupted by a 5 min period of reperfusion during which time the cuff was deflated. The control group had only a deflated cuff placed on the upper arm for 30 min. MAIN OUTCOME MEASURES: Serum troponin I concentration was measured before surgery and at 6, 12, 24, 48, and 72 h postoperatively. The cardiac function of all patients was followed postoperatively. RESULTS: Troponin I concentration was reduced in the RIPC group (398.7±179.3 µg/l) compared with the control group (708.4±242.5 µg/l). Mean difference was 309.7±50.8 (95% CI 210.1 to 409.3, p<0.0001). A greater improvement in postsurgical cardiac function was noted in the RIPC group than in the control group. CONCLUSIONS: These data indicate that RIPC reduces myocardial injury and improves cardiac function in patients undergoing heart valve surgery. TRIAL REGISTRATION NUMBER: NCT01175681.