Superoxide dismutase does not cause scar thinning after myocardial infarction

Previous studies demonstrated that treatment with superoxide dismutase, a scavenger of superoxide anions, limits the extent of myocardial injury in a canine preparation of regional myocardial ischemia and reperfusion. Little is known, however, about the effects of superoxide dismutase on the healing of a myocardial infarct. Therefore, this study was performed to determine whether treatment with superoxide dismutase during myocardial ischemia impairs formation of scar tissue after infarction. Dogs received 2 hour infusions of superoxide dismutase or albumin (controls) by way of the left atrium beginning 15 minutes before and ending 15 minutes after a 90 minute occlusion of the left circumflex coronary artery. Six weeks later the animals were killed. Two-dimensional echocardiography was performed before surgery and before induced death. Wall thickening in the central ischemic zone was decreased at 6 weeks compared with baseline studies (p less than 0.05), but the decrease was similar for both groups. The hydroxyproline concentrations (microgram/mg dry weight) of the scar tissue in the superoxide dismutase and control groups, respectively, were 35.3 +/- 3.8 and 28.7 +/- 5.0 (p less than 0.05). The ratios of the scar thickness to normal wall thickness were superoxide dismutase 0.91 +/- 0.03 and control 0.89 +/- 0.03 (p greater than 0.05). Thus, superoxide dismutase had no adverse effect on wall thickening or scar formation assessed 6 weeks after myocardial infarction, and may be useful to limit oxygen radical-mediated damage during reperfusion of the ischemic myocardium.     

Evidence for two distinct phenotypes of chronic kidney disease in individuals with type 1 diabetes mellitus

Aims/hypothesis

In a retrospective, observational, cross-sectional, single-centre study, we assessed the prevalence and correlates of different CKD phenotypes (with and without albuminuria) in a large cohort of patients of white ethnicity with type 1 diabetes.

Methods

From 2001 to 2009, 408 men and 369 women with type 1 diabetes (age 40.2 ± 11.7 years, diabetes duration 19.4 ± 12.2 years, HbA_1c 7.83 ± 1.17% [62.0 ± 12.9 mmol/mol]) were recruited consecutively. Albumin-to-creatinine ratio (ACR) and eGFR (Modification of Diet in Renal Disease) were obtained for all individuals, together with CKD stage. Diabetic retinopathy and peripheral polyneuropathy were detected in 41.5% and 8.1%, respectively, and cardiovascular disease (CVD) occurred in 8.5%. Adjudications of CKD phenotype were made by blinded investigators.

Results

Normo- (ACR <3.4), micro- (ACR 3.4–34) or macroalbuminuria (ACR ≥34 mg/mmol) were present in 91.6%, 6.4% and 1.9% of individuals, respectively. eGFR categories 1 (≥90 ml min^?1 [1.73 m]^?2), 2 (60–89 ml min^?1 [1.73 m]^?2) and 3 (<60 ml min^?1 [1.73 m]^?2) were present in 57.3%, 39.0% and 3.7%, respectively. The majority of participants had no CKD (89.4%), while stages 1–2 and ≥3 CKD were detected in 6.8% and 3.7%, respectively. The albuminuric (Alb⁺) and non-albuminuric (Alb^?) phenotypes were present in 12 (41.4%) and 17 (58.6%) individuals with stage ≥3 CKD, respectively. Individuals with an ACR <3.4 mg/mmol were subdivided into those with normal albuminuria (<1.1 mg/mmol; 77.2%) and mildly increased albuminuria (1.1–3.4 mg/mmol; 14.4%), and individuals with stage 2 CKD were subdivided into those with eGFR 75–89 ml min^?1 [1.73 m]^?2 and 60–74 ml min^?1 [1.73 m]^?2. ACR <3.4 mg/mmol (88.7%) and even <1.1 mg/mmol (70.4%) were common in individuals with eGFR 60–74 ml min^?1 [1.73 m]^?2. The prevalence of ACR <1.1 mg/mmol was lower but still significant (34.5%) in those with stage ≥3 CKD. In logistic regression analysis, stages 1–2 and ≥3 CKD were independently associated with age, HbA_1c, γ-glutamyltransferase, fibrinogen, hypertension, but not with sex, BMI, smoking, HDL-cholesterol or triacylglycerol. Inclusion of advanced retinopathy removed HbA_1c from the model. The CKD Alb⁺ phenotype correlated with diabetes duration, HbA_1c, HDL-cholesterol, fibrinogen and hypertension, while the CKD Alb^? phenotype was associated with age and hypertension, but not with diabetes duration, HbA_1c and fibrinogen.

Conclusions/interpretation

The Alb^? CKD phenotype is present in a significant proportion of individuals with type 1 diabetes supporting the hypothesis of two distinct pathways (Alb⁺ and Alb^?) of progression towards advanced kidney disease in type 1 diabetes. These are probably distinct pathways as suggested by different sets of covariates associated with the two CKD phenotypes.

     

Time-dependent changes in canine cardiac mitochondrial function and ultrastructure resulting from coronary occlusion and reperfusion

Summary Time-dependent changes in mitochondrial function and structure resulting from 1 hr of left circumflex coronary artery occlusion followed by 2 to 24 hrs of reperfusion were examined. These changes were correlated with changes in myocardial ultrastructure, tissue water content, infarct size and mitochondrial calcium content. The heart was removed after different periods of reperfusion, and mitochondria were isolated from ischemic and nonischemic regions of the left ventricle. Tissue samples from ischemic and nonischemic myocardium also were taken for electron microscopy and tissue water content determinations. Infarct size was measured by the nitroblue tetrazolium staining method. Oxygen consumption by mitochondria isolated from ischemic and nonischemic myocardium was measuredin vitro. Mitochondria from ischemic myocardium showed time-dependent decreases in rates of oxygen consumption and tightness of coupling. Electron microscopy revealed progressive ultrastructural deterioration in ischemic myocardium, including accumulation of calcium deposits within mitochondria, a finding corroborated by elevated concentrations of calcium in mitochondria isolated from the same area. Tissue wet-to-dry weight ratios were increased significantly in ischemic myocardium. A small, but significant, decrease in respiratory function was observed in mitochondria isolated from nonischemic myocardium several hrs after reperfusion; however, nomal respiration was observed 24 hrs after release of occlusion. This latter observation indicates that the nonischemic zone also is affected by regional ischemia. The results obtained indicate that temporary left circumflex artery occlusion and reperfusion result in progressively decreasing mitochondrial function and structure within the ischemic myocardium, and that these changes are accompanied by cellular electrolyte alterations.

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Zeitabhängige Veränderungen von Funktion und Ultrastruktur der Mitochondrien nach Koronarverschluß und Reperfusion beim Hundeherzen

Zusammenfassung Untersucht wurden zeitabhängige Veränderungen in Struktur und Funktion der Mitochondrien, die durch einstündigen Verschluß und 2- bis 24stündige Reperfusion des Ramus circumflexus der linken Koronararterie erzeugt wurden. Diese Veränderungen wurden mit Veränderungen der myokardialen Ultrastruktur, dem Wassergehalt des Gewebes, der Infarktgröße und dem mitochondrialen Calciumgehalt korreliert. Das Herz wurde nach verschiedenen Reperfusionszeiten entnommen und die Mitochondrien aus ischämischen und nichtischämischen Gebieten des linken Ventrikels isoliert. Ebenso wurden Gewebeproben von ischämischem und nichtischämischem Myokard für Elektronenmikroskopie und Bestimmung des Wassergehaltes des Gewebes entnommen. Die Infarktgröße wurde durch die Anfärbung mit Nitroblau-Tetrazolium bestimmt. Der Sauerstoffverbrauch der Mitochondrien aus ischämischem und nichtischämischem Myokard wurdein vitro gemessen. Mitochondrien aus ischämischem Myokard zeigten eine zeitabhängige Abnahme des Sauerstoffverbrauchs und seiner Bindung an die Phosphorylierung von ADP. Die Elektronenmikroskopie zeigte eine fortschreitende Zerstörung der Ultrastruktur im ischämischen Myokard, einschließlich einer Zunahme der Calciumablagerungen in Mitochondrien, was mit erhöhten Calciumkonzentrationen in Mitochondrien aus dem gleichen Gebiet übereinstimmte. Im ischämischen Myokard war die Relation Feuchtgewicht/Trockengewicht signifikant erhöht. Eine geringe, aber signifikante Abnahme der Atmung wurde in Mitochondrien, die nach einigen Stunden Reperfusion aus nichtischämischem Myokard isoliert worden waren, beobachtet; aber nach 24 h Reperfusion fand sich normale Atmung. Letzteres weist darauf hin, daß auch das nichtischämische Gebiet von der regionalen Ischämie betroffen ist. Die Ergebnisse zeigen, daß vorübergehender Verschluß des Ramus circumflexus der linken Koronararterie und Reperfusion zu fortschreitender Zerstörung mitochondrialer Funktion und Struktur führen und daß diese Veränderungen von Änderungen des Electrolytstatus der Zelle begleitet werden.

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With 5 figures and 2 tables

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This study was supported in part by U.S.P.H.S. Grant #HL-19782-03 and by Grants-in-Aid from the American and Michigan Heart Associations.

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Pre-doctoral Fellow supported by U.S.P.H.S. Training Grant #GM-00198-19.     

Inhibition of platelet-activating factor fails to limit ischemia and reperfusion-induced myocardial damage.

To determine the role of platelet-activating factor (1-O-hexa-decyl-2-acetyl-sn-glyceryl-phosphoryl-choline, PAF) in myocardial ischemic and reperfusion-induced injury, the effects of a PAF receptor antagonist (WEB 2086) were studied in an anesthetized canine model of ischemia (90 min) and reperfusion (6 h). Thirty minutes after onset of ischemia, WEB 2086 was administered as a bolus (20 mg/kg intravenously, i.v.) followed by a continuous 6-h infusion (10 mg/kg/h i.v.). Controls received vehicle alone (0.9% saline). Platelet aggregation was studied at baseline and at 1, 2, 4, and 6 h of drug administration and at the end of the reperfusion period. WEB 2086 treatment did not significantly affect platelet aggregation stimulated by ADP or arachidonic acid (AA). After 1 h of drug infusion, the ex vivo aggregatory response to exogenous (200 nM) PAF was ablated in WEB 2086-treated animals. WEB 2086 administration did not affect heart rate (HR) or mean arterial blood pressure (MAP) during the occlusion or reperfusion phases. During reperfusion of the ischemic tissue, left circumflex coronary artery (LCX) blood flow of WEB 2086-treated animals increased (p < 0.05) above control value. The area of the left ventricle at risk of infarct was not different between control and WEB 2086-treated groups. Infarct size was not significantly reduced in WEB 2086-treated animals. The results of our investigation using a 90-min ischemic period followed by 6-h reperfusion show that pharmacologic antagonism of PAF by WEB 2086 does not protect the heart against ischemia and reperfusion-induced injury.     

Assessment of clinical significance of anti-Ge in an untransfused man

A 19-year-old, untransfused Melanesian man from Papua New Guinea was admitted to the hospital for repair of an atrial septal defect. His serum contained an alloantibody that reacted strongly on the indirect antiglobulin test and was identified as anti-Ge. Gerbich-negative blood was transfused following urgent surgery. A 51Cr red cell survival study performed 2 weeks after surgery yielded zero survival of Gerbich-positive cells after 24 hours. A monocyte-driven, antibody-dependent, cell-mediated cytotoxicity assay performed on both pretransfusion and posttransfusion serum samples and on concentrated serum showed less than 1 percent specific lysis of Gerbich-positive cells. This did not correlate with the indication of clinical significance predicted by the 51Cr study. Red cell adherence and phagocytosis, not evident in a monocyte monolayer assay using native serum, were demonstrable in 16 percent of monocytes by the use of concentrated serum.     

Intimatan prevents arterial and venous thrombosis in a canine model of deep vessel wall injury

Resistance of fibrin-bound thrombin to inactivation by the heparin/antithrombin III complex is considered a limitation in the use of heparin as an antithrombotic agent. Intimatan (dermatan 4,6-di-O-sulfate) is a heparin cofactor II agonist that inhibits both free and bound forms of thrombin. The present study examines the hypothesis that Intimatan prevents thrombotic occlusion in response to vascular wall injury in a canine model of carotid artery/jugular vein thrombosis. The left carotid artery and right jugular vein served as vehicle-treated control vessels, whereas the right carotid artery and left jugular vein were subjected to electrolytic injury after administration of Intimatan (9 mg/kg bolus + 300 microg/kg/min infusion, i.v.) or dalteparin (Fragmin) (400 IU/kg, s.c.). Intimatan significantly increased time to carotid artery (226.0 +/- 14.0 min) and jugular vein (240.0 +/- 0.0 min) thrombosis, compared with control vessels (carotid artery, 87.1 +/- 7.9 min; jugular vein, 60.6 +/- 7.4 min). Vessel patency was maintained in eight of eight jugular veins and seven of eight carotid arteries during treatment with Intimatan. Dalteparin significantly increased time to carotid artery thrombosis (122.1 +/- 17.5 min) compared with control (64.3 +/- 8.2 min), but did not change the time to thrombosis in the jugular vein. Only one carotid artery remained patent at the end of the dalteparin protocol. The two drugs produced minimal increases in bleeding times, and Intimatan increased the activated partial thromboplastin time above that observed with dalteparin. The results demonstrate that Intimatan is effective in preventing occlusive arterial and venous thrombosis in an experimental model of deep vascular wall injury.