Grace Davis-Gorman

Thrombin activity and platelet microparticle formation are increased in type 2 diabetic platelets: a potential correlation with caspase activation.

Diabetics suffer from many complications including a prothrombotic condition. Activated platelet membrane provides an anchor, phosphatidylserine, for the attachment of the prothrombinase complex, which allows increased thrombin formation. This study aimed to further elucidate the interrelationship between coagulation proteins and activated platelets in type 2 diabetic blood. We found that there was a significant increase (30 x) in thrombin activity in the type 2 diabetic (ZDF) blood as compared to age-matched (ZL) controls (p<0.001). There was also a significant increase in the number of platelet microparticles in the type 2 diabetic rat compared to the lean control (p<0.001). Further, there were significant increases in caspase-3, -6, and -8 activities in the type 2 diabetic rats as compared to the lean controls (p<0.05). The combination of increased thrombin activity, increased PMP formation and increased caspase activity may contribute to the hypercoagulability of the diabetic blood. These results give more insight into the mechanisms underlying the interrelationship between diabetic platelets and coagulation proteins causing a prothrombotic condition in this patient population at increased risk from thromboembolic events.

Diabetes enhances leukocyte accumulation in the coronary microcirculation early in reperfusion following ischemia

BACKGROUND Diabetic hearts are particularly vulnerable to ischemia-reperfusion injury. For leukocytes to participate in ischemia-reperfusion injury, they must first sequester in the microcirculation. The aim of this study was to determine, by direct observation, if early leukocyte deposition was increased in the diabetic coronary microcirculation early in reperfusion following myocardial ischemia. METHODS Non-diabetic and streptozotocin (STZ)-induced diabetic rat hearts, subjected to 30 min of 37 degrees C, no-flow ischemia, were initially reperfused with blood containing labeled leukocytes. The deposition of fluorescent leukocytes in coronary capillaries and venules was directly visualized and recorded using intravital fluorescence microscopy. In addition, flow cytometry was used to measure CD11b adhesion molecule expression on polymorphonuclear (PMN) leukocytes from non-diabetic and STZ-diabetic rats. RESULTS In the non-diabetic, control hearts, early in reperfusion, leukocytes trapped in coronary capillaries and adhered to the walls of post-capillary venules. In the diabetic hearts, leukocyte trapping in capillaries and adhesion to venules were both significantly increased (P<0.05). PMN CD11b expression was also significantly increased in the diabetic blood compared to the non-diabetic blood (P<0.05). CONCLUSIONS Early in reperfusion following myocardial ischemia, leukocytes rapidly accumulate in greater numbers in the coronary microcirculation of the diabetic heart by both trapping in coronary capillaries and by adhering to venules. The enhanced retention of leukocytes in the diabetic coronary microcirculation increases the likelihood of inflammation-mediated reperfusion injury and may explain, in part, the poor recovery of diabetic hearts from an ischemic event.

Proinflammatory cytokines are increased in type 2 diabetic women with cardiovascular disease

Diabetics have a much greater morbidity and mortality due to cardiovascular disease (CVD) than nondiabetics. Furthermore, diabetic women have a 3.8-fold greater risk for CVD compared to diabetic men. Inflammation is now considered a risk factor for CVD and it has been demonstrated that inflammation also plays a role in diabetes. One component of inflammation that has reported to be increased in patients with diabetes only and CVD only are proinflammatory cytokines, particularly interleukin-6 (IL-6), tumor necrosis factor (TNF-alpha), and interleukin-1 (IL-1beta). This study was performed to test the hypothesis that these proinflammatory cytokines were increased in women with CVD and further increased in diabetic women with CVD compared to nondiabetic women with CVD and healthy age-matched controls. We found that IL-6 was increased in diabetic women with CVD compared to healthy age-matched controls (1.41 = 0.48 to 0.33 +/- 0.06 pg/ml, P < .05). IL-6 was also increased in diabetic women without CVD compared to healthy age-matched controls, but not significantly (0.96 +/- 0.27 to 0.33 +/- 0.06 pg/ml). We found that TNF-alpha was increased in diabetic women with and without CVD compared to healthy age-matched controls, but not significantly (4.53 +/- 1.38 to 3.93 +/ -0.53 to 2.33 +/- 0.89 pg/ml). IL-1beta was not significantly different among any of the four groups of women. These results indicate that both IL-6 and TNF-alpha are chronically increased in diabetic women with and without CVD compared to nondiabetic women. The additive concentration of cytokines in diabetes and CVD suggests a common inflammatory state in both diabetes and CVD.

Glucagon-like peptide-1 (GLP-1), immediately prior to reperfusion, decreases neutrophil activation and reduces myocardial infarct size in rodents.

Glucagon-like peptide-1 (GLP-1) is an incretin that has glucoregulatory effects as well as protective effects in a variety of tissues, including the heart. We hypothesized that GLP-1 may have a direct effect on neutrophils (PMNs) after myocardial ischemia, to ameliorate reperfusion injury. Deeply anesthetized Sprague-Dawley rats underwent 30 min of left coronary artery occlusion followed by 120 min of reperfusion. Immediately prior to reperfusion, rats were treated with either GLP-1 (human rGLP-1, 30 pM/kg/min) or PBS as placebo. GLP-1 significantly decreased myocardial infarct size [73.2±11.7% INF/AAR in PBS (n=4) vs. 15.7 ±5.52% INF/AAR in GLP-1-treated animals (n=5),  p<0.05], PMN activation in blood in vivo (fMLP-stimulated CD11b surface expression: PBS 2.78±1.14 vs. GLP-1 1.7±0.21, TFI, p<0.05), and accumulation in myocardium (PBS: 6.52±0.31 vs. GLP-1: 4.78±0.90, n=4-6 animals/group, p<0.05). In addition, we found that GLP-1 mitigated PMN CD11b surface expression in whole rat blood in vitro, an effect that was abolished by GLP-1 receptor blockade (PBS 6.52±0.31 vs. GLP-1 4.78±0.90, TFI, p<0.05). These findings suggest that one mechanism by which GLP-1 decreases reperfusion injury may be the attenuation of PMN-mediated reperfusion injury.

Effects of exercise training on responses to central injection of CRF and noise stress.

The purpose of this study was to test the hypothesis that the cardiovascular and sympathoadrenal responses to acute environmental stress are attenuated by exercise training. Furthermore, we tested the hypothesis that the cardiovascular and sympathoadrenal responses to intracerebroventricular (ICV) administration of corticotropin-releasing factor (CRF) would be attenuated by training. Conscious, unrestrained, male Sprague-Dawley rats assigned to either a treadmill trained (16-26 m/min, 30-60 min/day, 5 days/week) or nontrained (16-26 m/min, 10 min/day, 1 day/week) group were studied. After 8-10 weeks of training, maximal oxygen uptake was significantly higher in the trained (108 +/- 3 ml/kg/min) vs. the nontrained (94 +/- 4 ml/min/kg) group. There were no significant differences in baseline mean arterial pressure, heart rate and plasma catecholamine levels associated with training. Trained rats exhibited significantly attenuated elevations in arterial pressure (20 +/- 3 vs. 36 +/- 2 mmHg for nontrained) and heart rate (-3 +/- 3 vs. 12 +/- 5 beats/min for nontrained) in response to acute noise stress. Twenty minutes after ICV administration of CRF, blood pressure (trained = 119 +/- 2 mmHg, nontrained = 127 +/- 2 mmHg), heart rate (trained = 408 +/- 8 beats/min, nontrained = 424 +/- 10 beats/min), plasma norepinephrine levels (trained = 757 +/- 54 pg/ml, nontrained = 775 +/- 100 pg/ml) and plasma epinephrine levels (trained = 266 +/- 29 pg/ml, nontrained = 225 +/- 42 pg/ml) were significantly elevated in both trained and nontrained groups. CRF-induced elevations of blood pressure, but not heart rate or plasma catecholamine levels, were significantly attenuated in the trained group.(ABSTRACT TRUNCATED AT 250 WORDS)

Complement inhibition reduces injury in the type 2 diabetic heart following ischemia and reperfusion

Chronic inflammation exacerbates the cardiovascular complications of diabetes. Complement activation plays an important role in the inflammatory response and is known to be involved in ischemia-reperfusion (I/R) injury in the nondiabetic heart. The purpose of this study was to determine if increased complement deposition explains, in part, the increased severity of neutrophil-mediated I/R injury in the type 2 diabetic heart. Nondiabetic Zucker lean control (ZLC) and Zucker diabetic fatty (ZDF) rats underwent 30 min of coronary artery occlusion followed by 120 min of reperfusion. Another group of ZDF rats was treated with the complement inhibitor FUT-175 before reperfusion. Left ventricular (LV) tissue samples were stained for complement deposition and neutrophil accumulation following reperfusion. We found significantly more complement deposition in the ZDF LV compared with the ZLC (P < 0.05), and complement deposition was associated with significantly greater neutrophil accumulation. In whole blood samples taken preischemia and at 120 min reperfusion, neutrophils exhibited significantly more CD11b expression in the ZDF group compared with the ZLC group (P < 0.05). Furthermore, intracellular adhesion molecule (ICAM)-1 expression following I/R was increased significantly in ZDF hearts compared with ZLC hearts (P < 0.001). These results indicate that, in the ZDF heart, increased ICAM-1 and polymorphonuclear neutrophil (PMN) CD11b expression play a role in increasing PMN accumulation following I/R. The infarct size of the ZDF was significantly greater than ZLC (P < 0.05), and treatment with FUT-175 significantly decreased infarct size, complement deposition, and PMN accumulation in the diabetic heart. These findings indicate an exacerbated inflammatory response in the type 2 diabetic heart that contributes to the increased tissue injury observed following ischemia and reperfusion.

The Contribution of Mannose Binding Lectin to Reperfusion Injury after Ischemic Stroke

After complement system (CS) activation, the sequential production of complement products increases cell injury and death through opsonophagocytosis, cytolysis, adaptive, and inflammatory cell responses. These responses potentiate cerebral ischemia-reperfusion (IR) injury after ischemic stroke and reperfusion. Activation of the CS via mannose binding lectin (MBL)-initiated lectin pathway is known to increase tissue damage in response to IR in muscle, myocardium and intestine tissue. In contrast, the contribution of this pathway to cerebral IR injury, a neutrophil-mediated event, is less clear. Therefore, we investigated the potential protective role of MBL deficiency in neutrophil-mediated cerebral injury after IR. Using an intraluminal filament method, neutrophil activation and cerebral injury were compared between MBL-deficient and wild type C57Bl/6 mice subjected to 60 minutes of MCA ischemia and reperfusion. Systemic neutrophil activation was not decreased in MBL-deficient animals after IR. In MBL-deficient animals, cerebral injury was significantly decreased only in the striatum (p < 0.05). Despite MBL deficiency, C3 depositions were evident in the injured hemisphere during reperfusion. These results indicate that while MBL deficiency results in a modest protection of a sub-cortical brain region during IR, redundant complement pathway activation may overwhelm further beneficial effects of MBL deficiency during reperfusion.

Exaggerated neutrophil-mediated reperfusion injury after ischemic stroke in a rodent model of type 2 diabetes.

Please cite this paper as: Ritter, Davidson, Henry, Davis‐Gorman, Morrison, Frye, Cohen, Chandler, McDonagh and Funk (2011). Exaggerated Neutrophil‐Mediated Reperfusion Injury after Ischemic Stroke in a Rodent Model of Type 2 Diabetes. Microcirculation 18(7), 552–561.

Acute cardiovascular effects of firefighting and active cooling during rehabilitation.

Objectives: To determine the cardiovascular and hemostatic effects of fire suppression and postexposure active cooling. Methods: Forty-four firefighters were evaluated before and after a 12-minute live-fire drill. Next, 50 firefighters performing the same drill were randomized to undergo postfire forearm immersion in 10°C water or standard rehabilitation. Results: In the first study, heart rate and core body temperature increased and serum C-reactive protein decreased but there were no significant changes in fibrinogen, sE-selectin, or sL-selectin. The second study demonstrated an increase in blood coagulability, leukocyte count, factors VIII and X, cortisol, and glucose, and a decrease in plasminogen and sP-selectin. Active cooling reduced mean core temperature, heart rate, and leukocyte count. Conclusions: Live-fire exposure increased core temperature, heart rate, coagulability, and leukocyte count; all except coagulability were reduced by active cooling.

Inhibition of platelet GPIIb–IIIa and P-selectin expression by aspirin is impaired by stress hyperglycemia

Increased aspirin resistance may contribute to the increase in thrombotic events observed in patients with type 2 diabetes. In this study, we examined if acute exposure to increased plasma glucose impaired the inhibitory effects of aspirin on platelet activation. Whole-blood samples were incubated with 100 (euglycemia), 200, 300, and 600 mg/dl glucose followed by incubation with aspirin [acetylsalicylic acid (ASA)]. Using flow cytometry, GPIIb-IIIa and P-selectin were analyzed in unstimulated and arachidonic acid (AA)-stimulated platelets. In euglycemic blood, AA caused a significant increase in platelet GPIIb-IIIa expression [unstimulated: 59.5+/-8.2 total fluorescence intensity (TFI), AA stimulated: 319.6+/-42.7 TFI, P=.002] and P-selectin (4.4+/-0.7 and 179.5+/-38.5 TFI, P<.001). In vitro, ASA significantly inhibited both GPIIb-IIIa expression (36.5%) and P-selectin expression (81%; P<.005). However, increased blood glucose (200 mg/dl) significantly impaired the inhibitory effect of ASA (84% for GPIIb-IIIa, P<.005; 48% for P-selectin, P=NS). Increasing glucose to 600 mg/dl completely overwhelmed the inhibitory effect of ASA. A statistically significant interaction between glucose concentration and ASA dose was found (P<.001 for GPIIb-IIIa and P=.004 for P-selectin). In vitro, concentration-dependent stress hyperglycemia significantly impaired the inhibitory effects of aspirin on human platelet GPIIb-IIIa and P-selectin expression. Under acute hyperglycemic conditions, the effectiveness of ASA to inhibit platelets via the AA-activation pathway may be significantly reduced.