The central role of the neutrophil in ischaemic/reperfusion injury

Inadequate blood flow to an organ, ischaemia, may lead to both local and remote tissue injury characterized by oedema, increased microvascular permeability to protein and degradation of connective tissue components. This damage is probably caused by the accumulation and inappropriate activation of neutrophils which occurs when the tissue is reperfused. To test this hypothesis a number of in vitro models of the sequential stages of ischaemia/reperfusion injury were examined. Methods were initially developed to examine the adhesion of neutrophils to monolayers of a cultured endothelial cell line (ECV304) after periods of hypoxia and reoxygenation. Neutrophil migration in response to factors secreted by the treated endothelial cells was then assessed. The genesis of an inappropriate oxidative burst by the neutrophil upon exposure to endothelial chemoattractants and adhesion molecules was also measured. Finally to appraise how tissue function might be affected by endothelial cell hypoxia the contractility of vascular smooth muscle was examined. Neutrophil adhesion to ECV304 cells, which had been hypoxic for 4 hours and then reoxygenated for 30 minutes, was significantly increased. This response was probably initiated by reactive oxygen species (ROS) generated by the endothelial cells. Blockage of their production by allopurinol reduced the heightened adhesion. Similarly removal of ROS by superoxide dismutase or catalase also attenuated adhesion. ROS generation in turn caused the release of a soluble factor (s) which induced a conformational change on the neutrophil surface allowing it to bind to the intercellular adhesion molecule 1 (ICAM-1) on the endothelial cell. Soluble factor (s) from hypoxia/reoxygenated endothelial cells also had a powerful neutrophil chemoattractant ability. When neutrophils were exposed to both hypoxic/reoxygenated endothelial cells and the soluble factor (s) released by them a large oxidative burst was elicited. This response was greatest immediately after reoxygenation and one hour later was diminishing suggesting at least one of the components involved was labile. Analysis of the supernatant from hypoxic/reoxygenated endothelial cell cultures and studies using inhibitors of secretion suggested platelet activating factor (PAF) may be a major component in this overall sequence of events. Lesser roles for IL-8, TNF and LTB4 were also suggested. The secretory products from hypoxia/reoxygenated endothelial cells also affected smooth muscle contractility having an anti-vasoconstrictor or relaxation property, similar to that exerted by PAF.

36 monocyte subpopulations counts and associations with global longitudinal strain in st-elevation myocardial infarction patients with normal ejection fraction

Introduction - Monocytes, with 3 different subsets, are implicated in the initiation and progression of the atherosclerotic plaque contributing to plaque instability and rupture. Mon1 are the “classical” monocytes with inflammatory action, whilst Mon3 are considered reparative with fibroblast deposition ability. The function of the newly described Mon2 subset is yet to be fully described. In PCI era, fewer patients have globally reduced left ventricular ejection fraction post infarction, hence the importance of studying regional wall motion abnormalities and deformation at segmental levels using longitudinal strain. Little is known of the role for the 3 monocyte subpopulations in determining global strain in ST elevation myocardial infarction patients (STEMI). Conclusion In patients with normal or mildly impaired EF post infarction, higher counts of Mon1 and Mon2 are correlated with GLS within 7 days and at 6 months of remodelling post infarction. Adverse clinical outcomes in patients with reduced convalescent GLS were predicted with Mon1 and Mon2 suggestive of an inflammatory role for the newly identified Mon2 subpopulation. These results imply an important role for monocytes in myocardial healing when assessed by subclinical ventricular function indices. Methodology - STEMI patients (n = 101, mean age 64 ± 13 years; 69% male) treated with percutaneous revascularisation were recruited within 24 h post-infarction. Peripheral blood monocyte subpopulations were enumerated and characterised using flow cytometry after staining for CD14, CD16 and CCR2. Phenotypically, monocyte subpopulations are defined as: CD14++CD16-CCR2+ (Mon1), CD14++CD16+CCR2+ (Mon2) and CD14+CD16++CCR2- (Mon3). Phagocytic activity of monocytes was measured using flow cytometry and Ecoli commercial kit. Transthoracic 2D echocardiography was performed within 7 days and at 6 months post infarct to assess global longitudinal strain (GLS) via speckle tracking. MACE was defined as recurrent acute coronary syndrome and death. Results - STEMI patients with EF ≥50% by Simpson’s biplane (n = 52) had GLS assessed. Using multivariate regression analysis higher counts of Mon1 and Mon 2 and phagocytic activity of Mon2 were significantly associated with GLS (after adjusting for age, time to hospital presentation, and peak troponin levels) (Table 1). At 6 months, the convalescent GLS remained associated with higher counts of Mon1, Mon 2. At one year follow up, using multivariate Cox regression analysis, Mon1 and Mon2 counts were an independent predictor of MACE in patients with a reduced GLS (n = 21)

Carbon monoxide inhibits inward rectifier potassium channels in cardiomyocytes

Reperfusion-induced ventricular fibrillation (VF) severely threatens the lives of post-myocardial infarction patients. Carbon monoxide (CO) - produced by haem oxygenase in cardiomyocytes - has been reported to prevent VF through an unknown mechanism of action. Here, we report that CO prolongs action potential duration (APD) by inhibiting a subset of inward-rectifying potassium (Kir) channels. We show that CO blocks Kir2.2 and Kir2.3 but not Kir2.1 channels in both cardiomyocytes and HEK-293 cells transfected with Kir. CO directly inhibits Kir2.3 by interfering with its interaction with the second messenger phosphatidylinositol (4,5)-bisphosphate (PIP 2). As the inhibition of Kir2.2 and Kir2.3 by CO prolongs APD in myocytes, cardiac Kir2.2 and Kir2.3 are promising targets for the prevention of reperfusion-induced VF. © 2014 Macmillan Publishers Limited. All rights reserved.

Monocyte subpopulation counts and associations with left ventricular ejection fraction post ST elevation myocardial infarction

Background: Monocytes are implicated in the initiation and progression of the atherosclerotic plaque contributing to plaque instability and rupture. Little is known about the role of the three phenotypically and functionally different monocyte subpopulations in determining ventricular remodelling following ST elevation myocardial infarction (STEMI). Mon1 are the ‘classical’ monocytes with inflammatory action, whilst Mon3 are considered reparative with fibroblast deposition ability. The function of the newly described Mon2 subset is yet to be fully described. Method: STEMI patients (n=196, mean age 62±13 years; 72% male) treated with percutaneous revascularization were recruited within the first 24 h post-infarction. Peripheral blood monocyte subpopulations were enumerated and characterised using flow cytometry after staining for CD14, CD16 and CCR2. Phenotypically, monocyte subpopulations are defined as: CD14++CD16-CCR2+ (Mon1), CD14++CD16+CCR2+ (Mon2) and CD14+CD16++CCR2- (Mon3) cells. Transthoracic 2D echocardiography was performed within 7 days and at 6 months post infarct to assess ventricular volumes, mass, systolic, and diastolic functions as well as strain and strain rate. Results: Using linear regression analysis higher counts for Mon1, and lower counts for Mon2 and Mon3 were significantly associated with the baseline left ventricular ejection fraction (LVEF) within 7 days post infarct (table 1). At 6 months post STEMI lower counts of Mon2 remained positively associated with a decrease in LVEF at completion of remodelling (p=0.002). Conclusion: Peripheral monocytes of all three subsets correlate with LVEF after a myocardial infarction. High counts of the inflammatory Mon1 are associated with the reduced baseline ejection fraction post infarction. After remodelling, the convalescent ejection fraction was independently predicted by monocyte subpopulation 2. As lower counts depicted negative ventricular remodelling, this suggests a possible myofibroblast deposition and angiogenesis role for the newly described intermediate monocyte subpopulation Mon2 as opposed to the previously anticipated inflammatory role.

Monocyte subpopulation counts and TNF alpha associations with clinical outcome post ST elevation myocardial infarction

Background Monocytes are implicated in the initiation and progression of the atherosclerotic plaque contributing to its instability and rupture. Although peripheral monocytosis has been related to poor clinical outcome post ST elevation myocardial infarction (STEMI), only scarce information is available of mechanisms of this association. Tumour necrosis factor alpha (TNFα) is a key cytokine in the acute phase inflammatory response, and it is predominantly produced by inflammatory macrophages. Little is known about TNFα association with circulating monocyte subpopulations post STEMI. Method A total of 142 STEMI patients (mean age 62±13 years; 72% male) treated with percutaneous revascularization were recruited with blood samples obtained within first 24 hours from the onset and on day 10-14. Peripheral blood monocyte subpopulations were enumerated and characterized using flow cytometry after staining for CD14, CD16 and CCR2 and were defined as: CD14++CD16-CCR2+ (Mon1), CD14++CD16+CCR+ (Mon2) and CD14+CD16++CCR2- (Mon3) cells. Plasma levels of TNFα were measured by enzyme-linked immunosorbent assay (ELISA, Peprotec system, UK). Major adverse cardiac events (MACE), defined as recurrent STEMI, new diagnosis of heart failure and death were recorded at follow up, mean of 164±134 days. Results TNFα levels were significantly higher 24 hours post STEMI, compared to day 14 (paired t-test, p <0.001) with day 1 levels weakly correlated with total monocyte count as well as Mon1 (Spearman’s correlation, r=0.19, p=0.02 and r=0.22, p=0.01, respectively). There was no correlation between TNFα and Mon2 or Mon3 subpopulations. TNFα levels were significantly higher in patients with a recorded MACE (n=28, Mann-Whitney test, p<0.001) (figure 1).⇓

Monocyte subset counts associations with left ventricular systolic function post ST elevation myocardial infarction

Background: Monocytes are implicated in the initiation and progression of theatherosclerotic plaque contributing to plaque instability and rupture. Little is knownof the role played by the 3 phenotypically and functionally different monocytesubpopulations in determining ventricular remodeling following ST elevation my-ocardial infarction (STEMI). Mon1 are "classical" inflammatory monocytes, whilstMon3 are considered reparative with fibroblast deposition ability. The function ofthe newly described Mon2 is yet to be elucidated. Method: STEMI patients (n=196, mean age 62±13 years; 72% male) treatedwith percutaneous revascularization were recruited within the first 24 hours. Pe-ripheral blood monocyte subpopulations were enumerated and characterizedusing flow cytometry after staining for CD14, CD16 and CCR2. Phenotypi-cally, monocyte subpopulations are defined as: CD14+CD16-CCR2+ (Mon1),CD14+CD16+CCR+ (Mon2) and CD14lowCD16+CCR2- (Mon3) cells. Transtho-racic 2D echocardiography was performed within 7 days and 6 months post infarctto assess ventricular volumes, mass, systolic, and diastolic functions. Results: Using linear regression analysis higher counts for Mon1, and lowercounts for Mon2 and Mon3 were significantly associated with the baseline leftventricular ejection fraction (LVEF) within seven days post infarction. At 6 monthspost STEMI lower counts of Mon2 remained positively associated with decreasedLVEF (p value= 0.002).Monocyte subsets correlation with LVEFMonocytes mean florescence Baseline left ventricular Left ventricular ejectionintensity (cells/μl) ejection fraction (%) fraction (%) at 6 months post infarctβ-value P-valueβ-value P-valueTotal Mon0.31 P<0.001 0.360.009Mon 10.019 0.020.070.62Mon 2−0.28 0.001 −0.420.002Mon 3−0.27 0.001 −0.180.21 Conclusion: Peripheral monocytes of all three subsets correlate with LVEF af-ter a myocardial infarction. High counts of the inflammatory Mon1 are associatedwith reduction in the baseline LVEF. Post remodelling, the convalescent EF wasindependently predicted by monocyte subpopulation 2. As lower counts depictednegative ventricular remodeling, this suggests a reparative role for the newly de-scribed Mon2, possibly via myofibroblast deposition and angiogenesis, in contrastto an anticipated inflammatory role.

Cell-specific effects of Nox2 on the acute and chronic response to myocardial infarction

BACKGROUND: Increased reactive oxygen species (ROS) production is involved in the process of adverse cardiac remodeling and development of heart failure after myocardial infarction (MI). NADPH oxidase-2 (Nox2) is a major ROS source within the heart and its activity increases after MI. Furthermore, genetic deletion of Nox2 is protective against post-MI cardiac remodeling. Nox2 levels may increase both in cardiomyocytes and endothelial cells and recent studies indicate cell-specific effects of Nox2, but it is not known which of these cell types is important in post-MI remodeling. METHODS AND RESULTS: We have generated transgenic mouse models in which Nox2 expression is targeted either to cardiomyocytes (cardio-Nox2TG) or endothelial cells (endo-Nox2TG). We here studied the response of cardio-Nox2TG mice, endo-Nox2TG mice and matched wild-type littermates (WT) to MI induced by permanent left coronary artery ligation up to 4weeks. Initial infarct size assessed by magnetic resonance imaging (MRI) and cardiac dysfunction were similar among groups. Cardiomyocyte hypertrophy and interstitial fibrosis were augmented in cardio-Nox2TG compared to WT after MI and post-MI survival tended to be worse whereas endo-Nox2TG mice showed no significant difference compared to WT. CONCLUSIONS: These results indicate that cardiomyocyte rather than endothelial cell Nox2 may have the more important role in post-MI remodeling.

In vivo characterization of myocardial tissue post myocardial infarction using combined fluorescence and diffuse reflectance spectroscopy

Accurately assessing the extent of myocardial tissue injury induced by Myocardial infarction (MI) is critical to the planning and optimization of MI patient management. With this in mind, this study investigated the feasibility of using combined fluorescence and diffuse reflectance spectroscopy to characterize a myocardial infarct at the different stages of its development. An animal study was conducted using twenty male Sprague-Dawley rats with MI. In vivo fluorescence spectra at 337 nm excitation and diffuse reflectance between 400 nm and 900 nm were measured from the heart using a portable fiber-optic spectroscopic system. Spectral acquisition was performed on (1) the normal heart region; (2) the region immediately surrounding the infarct; and (3) the infarcted region—one, two, three and four weeks into MI development. The spectral data were divided into six subgroups according to the histopathological features associated with various degrees/severities of myocardial tissue injury as well as various stages of myocardial tissue remodeling, post infarction. Various data processing and analysis techniques were employed to recognize the representative spectral features corresponding to various histopathological features associated with myocardial infarction. The identified spectral features were utilized in discriminant analysis to further evaluate their effectiveness in classifying tissue injuries induced by MI. In this study, it was observed that MI induced significant alterations (p < 0.05) in the diffuse reflectance spectra, especially between 450 nm and 600 nm, from myocardial tissue within the infarcted and surrounding regions. In addition, MI induced a significant elevation in fluorescence intensities at 400 and 460 nm from the myocardial tissue from the same regions. The extent of these spectral alterations was related to the duration of the infarction. Using the spectral features identified, an effective tissue injury classification algorithm was developed which produced a satisfactory overall classification result (87.8%). The findings of this research support the concept that optical spectroscopy represents a useful tool to non-invasively determine the in vivo pathophysiological features of a myocardial infarct and its surrounding tissue, thereby providing valuable real-time feedback to surgeons during various surgical interventions for MI.

In vivo tissue diagnosis for myocardial infarction using optical spectroscopy with novel spectral interpretation algorithms

In recent decades, the rapid development of optical spectroscopy for tissue diagnosis has been indicative of its high clinical value. The goal of this research is to prove the feasibility of using diffuse reflectance spectroscopy and fluorescence spectroscopy to assess myocardial infarction (MI) in vivo. The proposed optical technique was designed to be an intra-operative guidance tool that can provide useful information about the condition of an infarct for surgeons and researchers. ^ In order to gain insight into the pathophysiological characteristics of an infarct, two novel spectral analysis algorithms were developed to interpret diffuse reflectance spectra. The algorithms were developed based on the unique absorption properties of hemoglobin for the purpose of retrieving regional hemoglobin oxygenation saturation and concentration data in tissue from diffuse reflectance spectra. The algorithms were evaluated and validated using simulated data and actual experimental data. ^ Finally, the hypothesis of the study was validated using a rabbit model of MI. The mechanism by which the MI was induced was the ligation of a major coronary artery of the left ventricle. Three to four weeks after the MI was induced, the extent of myocardial tissue injury and the evolution of the wound healing process were investigated using the proposed spectroscopic methodology as well as histology. The correlations between spectral alterations and histopathological features of the MI were analyzed statistically. ^ The results of this PhD study demonstrate the applicability of the proposed optical methodology for assessing myocardial tissue damage induced by MI in vivo. The results of the spectral analysis suggest that connective tissue proliferation induced by MI significantly alter the characteristics of diffuse reflectance and fluorescence spectra. The magnitudes of the alterations could be quantitatively related to the severity and extensiveness of connective tissue proliferation.^

In Vivo Characterization of Myocardial Tissue Post Myocardial Infarction Using Combined Fluorescence and Diffuse Reflectance Spectroscopy

Accurately assessing the extent of myocardial tissue injury induced by Myocardial infarction (MI) is critical to the planning and optimization of MI patient management. With this in mind, this study investigated the feasibility of using combined fluorescence and diffuse reflectance spectroscopy to characterize a myocardial infarct at the different stages of its development. An animal study was conducted using twenty male Sprague-Dawley rats with MI. In vivo fluorescence spectra at 337 nm excitation and diffuse reflectance between 400 nm and 900 nm were measured from the heart using a portable fiber-optic spectroscopic system. Spectral acquisition was performed on - (1) the normal heart region; (2) the region immediately surrounding the infarct; and (3) the infarcted region - one, two, three and four weeks into MI development. The spectral data were divided into six subgroups according to the histopathological features associated with various degrees / severities of myocardial tissue injury as well as various stages of myocardial tissue remodeling, post infarction. Various data processing and analysis techniques were employed to recognize the representative spectral features corresponding to various histopathological features associated with myocardial infarction. The identified spectral features were utilized in discriminant analysis to further evaluate their effectiveness in classifying tissue injuries induced by MI. In this study, it was observed that MI induced significant alterations (p < 0.05) in the diffuse reflectance spectra, especially between 450 nm and 600 nm, from myocardial tissue within the infarcted and surrounding regions. In addition, MI induced a significant elevation in fluorescence intensities at 400 and 460 nm from the myocardial tissue from the same regions. The extent of these spectral alterations was related to the duration of the infarction. Using the spectral features identified, an effective tissue injury classification algorithm was developed which produced a satisfactory overall classification result (87.8%). The findings of this research support the concept that optical spectroscopy represents a useful tool to non-invasively determine the in vivo pathophysiological features of a myocardial infarct and its surrounding tissue, thereby providing valuable real-time feedback to surgeons during various surgical interventions for MI.

T1 mapping for assessment of myocardial injury and microvascular obstruction at one week post myocardial infarction

Acknowledgements This study was supported by a Medical Research Council UK grant (grant number G0800901), as a sub-study of Nitrites in Acute Myocardial Infarction. Thanks are due to Roger Staff, for invaluable advice regarding receiver operator characteristic analysis.

T1 mapping for assessment of myocardial injury and microvascular obstruction at one week post myocardial infarction

Acknowledgements This study was supported by a Medical Research Council UK grant (grant number G0800901), as a sub-study of Nitrites in Acute Myocardial Infarction. Thanks are due to Roger Staff, for invaluable advice regarding receiver operator characteristic analysis.

Association of increasing age with receipt of specialist care and long-term mortality in patients with non-ST elevation myocardial infarction

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