Neutrophil extracellular traps in ischemia-reperfusion injury-induced myocardial no-reflow: therapeutic potential of DNase-based reperfusion strategy.

Emerging evidence suggests a potential role of neutrophil extracellular traps (NETs) in linking sterile inflammation and thrombosis. We hypothesized that NETs would be induced during myocardial ischemia-reperfusion (I/R), and NET-mediated microthrombosis may contribute to myocardial "no-reflow". Male Wistar rats were randomly divided into I/R control, DNase (DNase I, 20 µg/rat), recombinant tissue-type plasminogen activator (rt-PA, 420 µg/rat), DNase + rt-PA, and sham control groups after 45-min myocardial ischemia. In situ NET formation, the anatomic "no re-flow" area, and infarct size were evaluated immediately after 3 h of reperfusion. Long-term left ventricular (LV) functional and histological analyses were performed 45 days after operation. Compared with the I/R controls, the DNase + rt-PA group exhibited reduced NET density [8.38 ± 1.98 vs. 26.86 ± 3.07 (per 200 × field), P < 0.001] and "no-flow" area (15.22 ± 0.06 vs. 34.6 ± 0.05%, P < 0.05) in the ischemic region, as well as reduced infarct size (38.39 ± 0.05 vs. 71.00 ± 0.03%, P < 0.001). Additionally, compared with the I/R controls, DNase + rt-PA treatment significantly ameliorated I/R injury-induced LV remodeling (LV ejection fraction: 64.22 ± 3.37 vs. 33.81 ± 2.98%, P < 0.05; LV maximal slope of the LV systolic pressure increment: 3,785 ± 216 vs. 2,596 ± 299 mmHg/s, P < 0.05). The beneficial effect was not observed in rats treated with DNase I or rt-PA alone. Our study provides evidence for the existence of NETs in I/R-challenged myocardium and confirms the long-term benefit of a novel DNase-based reperfusion strategy (DNase I + rt-PA), which might be a promising option for the treatment of myocardial I/R injury and coronary no-reflow.

Monocyte subsets and monocyte-platelet aggregates in patients with unstable angina.

Monocyte subsets and monocyte-platelet aggregates (MPAs) play important role in atherosclerosis and thrombosis. We aimed to determine their changes in patients with unstable angina (UA). In this cross-sectional case-control study, Global Registry of Acute Coronary Events (GRACE) score was determined in 95 UA patients without elevated troponin level. Thirty age-and-sex matched stable coronary heart disease (CHD) subjects served as control group. The classical (CD14++CD16-, Mon1), the intermediate (CD14++CD16+, Mon2) and the non-classical (CD14+CD16++, Mon3) monocytes, as well as subset-specific MPAs, were measured by flow cytometry. Compared with stable CHD patients, UA patients had increased Mon2 and Mon3 counts (all P < 0.001). For UA subjects, compared with GRACE score-determined low risk patients (GRACE score ≤108, n = 70), intermediate-to-high risk patients (GRACE score >108, n = 25) had higher counts of Mon2 and total MPAs, as well as Mon1- and Mon2-associated MPAs (all P < 0.001). Adjusted binary logistic regression analysis revealed that increased counts of Mon2 subset (for per 5 cells/µL increase, OR 1.186, 95% CI 1.044-1.347, P = 0.009), Mon2 MPAs (for per 5 cells/µL increase, OR 1.228, 95% CI 1.062-1.421, P = 0.006) and total MPAs (for per 5 cells/µL increase, OR 1.072, 95 % CI 1.010-1.137, P = 0.022) independently associated with GRACE score-determined intermediate-to-high risk UA patients. In UA patients with intermediate-to-high risk (determined by GRACE score), counts of Mon2 subset, Mon2-associated MPAs and total MPAs are increased, which are independent of traditional risk factors.

Decreased circulating microRNA-223 level predicts high on-treatment platelet reactivity in patients with troponin-negative non-ST elevation acute coronary syndrome.

To investigate the relationship between circulating microRNA 223 (miR-223) levels and clopidogrel responsiveness in patients with coronary heart disease. A total of 62 consecutive patients with troponin-negative non-ST elevation acute coronary syndrome (NSTE-ACS) scheduled for elective percutaneous coronary intervention were enrolled. The plasma circulating miR-223 levels were quantified by real-time PCR, and platelet reactivity was determined by platelet reactivity index (PRI), measured by vasodilator-stimulated phosphoprotein (VASP) phosphorylation flow cytometry after 300 mg (for at least 24 h) or 75 mg clopidogel (for at least 5 days) plus aspirin treatment. All subjects were dichotomized according to PRI median (normal-responders: PRI ≤ 56.3%, n = 31 and low-responders: PRI > 56.3%, n = 31). Compared with normal-responders, circulating miR-223 level was significantly decreased in low-responders (P = 0.007). In addition, miR-223 level was statistically correlated with PRI (Spearman r = -0.379, P = 0.002). Stepwise binary logistic regression analysis revealed that among factors that potentially influence platelet reactivity (CYP2C19*2/*3 loss-of-function genotypes, use of calcium channel blockers/proton-pump inhibitors, age, diabetes and smoking), decreased circulating miR-223 level was the only independent predictor for the presence of PRI-determined lower responders (OR 0.111, 95% CI 0.018-0.692, P = 0.019). Our data suggest that circulating miR-223 may serve as a novel biomarker for assessment of clopidogrel responsiveness in troponin-negative NSTE-ACS patients.

Discordance Between VASP Phosphorylation and Platelet Aggregation in Defining High On-Clopidogrel Platelet Reactivity After ST-Segment Elevation Myocardial Infarction.

To investigate potential clinical characteristics associated with discordance between platelet vasodilator-stimulated phosphoprotein phosphorylation (VASP-P) flow cytometry (FCM) assay and light transmission aggregometry (LTA) in defining high on-clopidogrel platelet reactivity (HPR) after ST-segment elevation myocardial infarction (STEMI). In this study, platelet responsiveness was measured by the above 2 methods simultaneously on day 1 and on day 6 of STEMI onset in 90 consecutive patients who underwent primary percutaneous coronary intervention. The FCM-derived platelet reactivity index and LTA-derived platelet aggregation rate were both significantly reduced after dual antiplatelet therapy on day 6. Multiple variable-adjusted logistic regression analysis revealed that smoking (odds ratio [OR]: 4.507, 95% confidence interval [CI]: 1.123-18.09, P = .034) and onset-to-admission time (per 1 hour increase, OR: 1.196, 95% CI: 1.023-1.398, P = .025) both were independent predictors for the discordance between the 2 methods. Additionally, improved correlation and concordance was observed in nonsmokers compared with smokers. Our data show that smoking and prolonged onset-to-admission time are associated with discordance between platelet VASP-P and LTA in defining HPR after STEMI, which should be considered when planning personalized antiplatelet therapy.

The influence of different anticoagulants and time-delayed sample processing and measurements on human monocyte subset and monocyte-platelet aggregate analyses.

BACKGROUND: Measuring human monocyte subsets (CD14++CD16-, CD14++CD16+, and CD14 + CD16++) and subset-specific monocyte-platelet aggregates (MPA) is vulnerable to analytical bias due to unavailability of a standardized methodology. We aimed to address this issue by focusing on the impacts of time-delayed sample processing and measurement between two commonly used anticoagulants. METHODS: Ethylenediaminetetraacetic acid (EDTA)- and sodium citrate (SC)-anticoagulated blood samples from 12 healthy donors were subject to either delayed (2-h delay, kept at 4°C) or immediate processing (without fixation) before four-color flow cytometry (FCM) analysis. RESULTS: In SC-anticoagulated samples, a 2-h delay in sample processing contributed to a significant decrease in CD14++CD16- monocyte percent and a reciprocal increase in CD14++CD16+ monocytes, as well as increases in all three subset-specific MPA. Similar slight, but non-significant changes were observed in EDTA-treated samples. In samples processed immediately and stored at 4°C, delayed measurement at 0, 1, 3, and 5 h after processing led to a time-dependent decrease in CD14++CD16- monocyte percent and a reciprocal increase in CD14++CD16+ subset in SC-treated, but not in EDTA-treated, samples. Moreover, a time-dependent increase in all three subset-specific MPA was observed in SC-treated samples, which, to a lesser extent, was only observed in CD14++CD16+ MPA in EDTA-treated samples after storage at 4°C for 3-5 h after processing. CONCLUSIONS: We recommend EDTA for anticoagulation. Additionally, sample should be stored at 4°C and processing and measuring should be performed within 2 h after harvest and 3 h after processing, respectively. © 2016 International Clinical Cytometry Society.

High-salt intake induced visceral adipose tissue hypoxia and its association with circulating monocyte subsets in humans.

OBJECTIVE: To investigate the feasibility of blood oxygen level dependent magnetic resonance imaging (BOLD-MRI) in evaluating human visceral adipose tissue (AT) oxygenation induced by salt loading/depletion and its association with changes in circulating monocyte subsets. METHODS: A dietary intervention study was performed in 23 healthy volunteers beginning with a 3-day usual diet followed by a 7-day high-salt diet (≥15 g NaCl/day) and a 7-day low-salt diet (≤5 g NaCl/day). BOLD-MRI was used to evaluate oxygenation in perirenal AT. RESULTS: Salt loading led to a consistent AT hypoxia (increase in the R2* signal, 25.2 ± 0.90 s(-1) vs. baseline 21.5 ± 0.71 s(-1) , P < 0.001) and suppression of circulating renin-angiotensin-aldosterone system (RAAS), as well as an expansion of the CD14++CD16+ monocytes and monocyte pro-inflammatory activation. In salt depletion phase, the hypoxic state of AT and the expanded CD14++CD16+ monocyte pool were regressed to baseline levels, accompanied by a rebound activation of RAAS. Moreover, AT oxygenation level was positively correlated with the CD14++CD16+ monocytes (r = 0.419, P < 0.001). CONCLUSIONS: This work provides proof-of-principle evidence supporting the feasibility of BOLD-MRI in monitoring visceral AT oxygenation in humans induced by dietary salt loading/depletion. In addition, the CD14++CD16+ monocytes may participate in the pathogenesis of high-salt intake induced AT hypoxia.

Temporal and spatial characterization of mononuclear phagocytes in circulating, lung alveolar and interstitial compartments in a mouse model of bleomycin-induced pulmonary injury.

The mononuclear phagocyte system, including circulating monocytes and tissue resident macrophages, plays an important role in acute lung injury and fibrosis. The detailed dynamic changes of mononuclear phagocytes in the circulating, lung alveolar and interstitial compartments in bleomycin-induced pulmonary injury model have not been fully characterized. The present study was designed to address this issue and analyzed their relationships with pulmonary pathological evolution after bleomycin challenge. A total of 100 male C57BL/6 mice were randomly divided to receive bleomycin (2.5mg/kg, n=50) or normal saline (n=50) via oropharyngeal approach, and were sacrificed on days 1, 3, 7, 14 and 21. Circulating monocyte subsets, polarization state of bronchoalveolar lavage fluid (BALF)-derived alveolar macrophages (AMφ) and lung interstitial macrophages (IMφ, derived from enzymatically digested lung tissue) were analyzed by flow cytometry. There was a rapid expansion of circulating Ly6C(hi) monocytes which peaked on day 3, and its magnitude was positively associated with pulmonary inflammatory response. Moreover, an expansion of M2-like AMφ (F4/80+CD11c+CD206+) peaked on day 14, and was positively correlated with the magnitude of lung fibrosis. The polarization state of IMφ remained relatively stable in the early- and mid-stage after bleomycin challenge, expect for an increase of M2-like (F4/80+CD11c-CD206+) IMφ on day 21. These results support the notion that there is a Ly6C(hi)-monocyte-directed pulmonary AMφ alternative activation. Our result provides a dynamic view of mononuclear phagocyte change in three compartments after bleomycin challenge, which is relevant for designing new treatment strategies targeting mononuclear phagocytes in this model.

Decreased platelet miR-223 expression is associated with high on-clopidogrel platelet reactivity.

OBJECTIVES: We aimed to investigate the relationship between platelet microRNA (miR-223 and miR-96) expression and clopidogrel responsiveness in patients with coronary heart disease (CHD). MATERIALS AND METHODS: A total of 33 consecutive non-diabetic CHD patients scheduled for percutaneous coronary intervention were enrolled. Platelet reactivity after clopidogrel loading dose (300 mg) was determined by two methods [platelet reactivity index (PRI), measured by vasodilator-stimulated phosphoprotein (VASP) phosphorylation flow cytometry and ADP-induced platelet aggregation (PAG), measured by light transmission aggregometry]. Total platelet RNA was isolated from purified platelets (CD45 magnetic bead negative selection) to quantify miR-223 and miR-96 expression by real-time PCR. RESULTS: All subjects were dichotomized according to PRI medians (normal-responders: PRI < 56.5%, n = 17 and low-responders: PRI > 56.5%, n = 16) and PAG medians (normal-responders: PAG < 43%, n = 17 and low-responders: PAG > 43%, n = 16). Compared with PRI-determined normal-responders, miR-223 expression, but not miR-96, was significantly decreased in low-responders (P = 0.037). No differential expression of miR-223 and miR-96 was observed via PAG determination between normal- and low-responders. In addition, miR-223 expression, but not miR96, was statistically correlated with PRI (Spearman r = -0.403, P = 0.020). Stepwise binary logistic regression analysis revealed that among factors that potentially influence platelet reactivity (CYP2C19*2 loss-of-function genotypes, use of calcium channel blockers/proton-pump inhibitors, age, obesity, smoking and platelet microRNAs), decreased miR-223 expression was the only independent predictor associated with the presence of PRI-determined low responders to clopidogrel (OR 0.189, 95% CI 0.043 to 0.836, P = 0.028). CONCLUSIONS: The present work identifies decreased platelet miR-223 expression as a novel mechanism involved in blunted platelet response to clopidogrel in a Chinese population.

Modulation of mononuclear phagocyte inflammatory response by liposome-encapsulated voltage gated sodium channel inhibitor ameliorates myocardial ischemia/reperfusion injury in rats.

BACKGROUND: Emerging evidence shows that anti-inflammatory strategies targeting inflammatory monocyte subset could reduce excessive inflammation and improve cardiovascular outcomes. Functional expression of voltage-gated sodium channels (VGSCs) have been demonstrated in monocytes and macrophages. We hypothesized that mononuclear phagocyte VGSCs are a target for monocyte/macrophage phenotypic switch, and liposome mediated inhibition of mononuclear phagocyte VGSC may attenuate myocardial ischemia/reperfusion (I/R) injury and improve post-infarction left ventricular remodeling. METHODOLOGY/PRINCIPAL FINDINGS: Thin film dispersion method was used to prepare phenytoin (PHT, a non-selective VGSC inhibitor) entrapped liposomes. Pharmacokinetic study revealed that the distribution and elimination half-life of PHT entrapped liposomes were shorter than those of free PHT, indicating a rapid uptake by mononuclear phagocytes after intravenous injection. In rat peritoneal macrophages, several VGSC α subunits (NaV1.1, NaV1.3, NaV1.4, NaV1.5, NaV1.6, NaV1.7, NaVX, Scn1b, Scn3b and Scn4b) and ß subunits were expressed at mRNA level, and PHT could suppress lipopolysaccharide induced M1 polarization (decreased TNF-α and CCL5 expression) and facilitate interleukin-4 induced M2 polarization (increased Arg1 and TGF-ß1 expression). In vivo study using rat model of myocardial I/R injury, demonstrated that PHT entrapped liposome could partially suppress I/R injury induced CD43+ inflammatory monocyte expansion, along with decreased infarct size and left ventricular fibrosis. Transthoracic echocardiography and invasive hemodynamic analysis revealed that PHT entrapped liposome treatment could attenuate left ventricular structural and functional remodeling, as shown by increased ejection fraction, reduced end-systolic and end-diastolic volume, as well as an amelioration of left ventricular systolic (+dP/dt max) and diastolic (-dP/dt min) functions. CONCLUSIONS/SIGNIFICANCE: Our work for the first time demonstrates the therapeutic potential of VGSC antagonism via liposome mediated monocyte/macrophage targeting in acute phase after myocardial I/R injury. These results suggest that VGSCs in mononuclear phagocyte system might be a novel target for immunomodulation and treatment of myocardial I/R injury.

Spironolactone attenuates bleomycin-induced pulmonary injury partially via modulating mononuclear phagocyte phenotype switching in circulating and alveolar compartments.

BACKGROUND: Recent experimental studies provide evidence indicating that manipulation of the mononuclear phagocyte phenotype could be a feasible approach to alter the severity and persistence of pulmonary injury and fibrosis. Mineralocorticoid receptor (MR) has been reported as a target to regulate macrophage polarization. The present work was designed to investigate the therapeutic potential of MR antagonism in bleomycin-induced acute lung injury and fibrosis. METHODOLOGY/PRINCIPAL FINDINGS: We first demonstrated the expression of MR in magnetic bead-purified Ly6G-/CD11b+ circulating monocytes and in alveolar macrophages harvested in bronchoalveolar lavage fluid (BALF) from C57BL/6 mice. Then, a pharmacological intervention study using spironolactone (20 mg/kg/day by oral gavage) revealed that MR antagonism led to decreased inflammatory cell infiltration, cytokine production (downregulated monocyte chemoattractant protein-1, transforming growth factor ß1, and interleukin-1ß at mRNA and protein levels) and collagen deposition (decreased lung total hydroxyproline content and collagen positive area by Masson' trichrome staining) in bleomycin treated (2.5 mg/kg, via oropharyngeal instillation) male C57BL/6 mice. Moreover, serial flow cytometry analysis in blood, BALF and enzymatically digested lung tissue, revealed that spironolactone could partially inhibit bleomycin-induced circulating Ly6C(hi) monocyte expansion, and reduce alternative activation (F4/80+CD11c+CD206+) of mononuclear phagocyte in alveoli, whereas the phenotype of interstitial macrophage (F4/80+CD11c-) remained unaffected by spironolactone during investigation. CONCLUSIONS/SIGNIFICANCE: The present work provides the experimental evidence that spironolactone could attenuate bleomycin-induced acute pulmonary injury and fibrosis, partially via inhibition of MR-mediated circulating monocyte and alveolar macrophage phenotype switching.