Donald A.G. Mickle

Resistin Promotes Endothelial Cell Activation Further Evidence of Adipokine-Endothelial Interaction

Background—Adipocyte-derived hormones may represent a mechanism linking insulin resistance to cardiovascular disease. In the present study, we evaluated the direct effects of resistin, a novel adipocyte-derived hormone, on endothelial activation. Methods and Results—Endothelial cells (ECs) were incubated with human recombinant resistin (10 to 100 ng/ML, 24 hours), and endothelin-1 (ET-1) release, ET-1 mRNA expression, and nitric oxide (NO) production were assessed. Transient transfection assays were used to evaluate the effects of resistin on transcription of human ET-1 gene promoter. Furthermore, the effects of resistin on AP-1–mutated ET-1 promoter were evaluated. The effects of resistin on expression of vascular cell adhesion molecule (VCAM-1) and monocyte chemoattractant chemokine (MCP-1) were studied in addition to CD40 receptor, CD40 ligand–induced MCP-1 expression, and tumor necrosis factor receptor–associated factor-3 (TRAF3), an inhibitor of CD40 signaling. Incubation of ECs with resistin resulted in an increase in ET-1 release and ET-1 mRNA expression, with no change in NO production. Whereas treatment with resistin resulted in an increase in ET-1 promoter activity, the AP-1–mutated promoter was inactive after resistin stimulation. Additionally, resistin-treated cells showed increased expression of VCAM-1 and MCP-1, with concomitant reductions in TRAF-3 expression. Resistin did not alter CD40 receptor expression; however, increased CD40 ligand induced MCP-1 production. Conclusions—The novel adipokine resistin exerts direct effects to promote EC activation by promoting ET-1 release, in part by inducing ET-1 promoter activity via the AP-1 site. Furthermore, resistin upregulates adhesion molecules and chemokines and downregulates TRAF-3, an inhibitor of CD40 ligand signaling. In this fashion, resistin may be mechanistically linked to cardiovascular disease in the metabolic syndrome.

Salvage of skeletal muscle with free radical scavengers

Extensive skeletal muscle necrosis may occur after prolonged ischemia to the lower extremity, with serious consequences both locally and systemically. The extent of necrosis is a combination of cellular damage that occurs during both the period of ischemia and the period of reperfusion. The purpose of this study was to reduce the extent of reperfusion-induced muscle necrosis by therapeutic interventions administered only during the initial period of reperfusion. Indeed, the pretreatment of patients who have an acute arterial occlusion is rarely possible and only interventions applicable to the reperfusion phase would be clinically relevant. By perfusing the isolated gracilis muscle in a controlled manner with reduced oxygen concentrations alone and in combination with free radical scavengers, we were able to reduce the extent of muscle necrosis. By means of controlled oxygen delivery alone, muscle necrosis was reduced from 87% +/- 8% in the control muscle to 67% +/- 9% (p less than 0.05) in the treated muscle. The combination of reduced oxygen delivery and free radical scavengers reduced necrosis from 78% +/- 8% in the control muscle to 53% +/- 7% (p less than 0.01) on the experimental side. We conclude that controlled oxygen delivery and free radical scavengers can reduce skeletal muscle necrosis occurring after prolonged normothermic ischemia.

Conjugated dienes in ischemic and reperfused myocardium: an in vivo chemical signature of oxygen free radical mediated injury

Free radical reactions have been suggested to be important events in the mechanism of myocardial injury during ischemia and reperfusion. Most of the in vivo evidence implicating free radical mediated events in the etiology of myocardial injury has been based on intervention studies which document the efficacy of oxygen free radical scavengers in improving function or tissue salvage. We have assayed free fatty acid oxidation products (hydroxy conjugated dienes) derived from myocardial phospholipid as chemical markers of oxidative injury. Biopsies, harvested from canine myocardium subjected to cardiopulmonary bypass with no aortic crossclamp (control), from pre-ischemic, end ischemic (at the end of 45 min of global normothermic ischemia induced by aortic crossclamp) and at 5, 10, 15, 30 and 60 mins of reperfusion were analyzed for hydroxy conjugated dienes using HPLC with structural confirmation by GC-MS. All biopsies were taken from non-necrotic myocardium as indicated by gross tetrazolium staining of myocardial cross sections. Trace levels of hydroxy conjugated dienes could be detected in the preischemic biopsies or control biopsies harvested from hearts subjected to cardiopulmonary bypass with no ischemia. At the end of 45 min ischemia, however, a significant increase in 18:2 and 20:4 hydroxy isomers was detected and confirmed by GC-MS (P less than 0.05 vs. control). After 5 mins of reperfusion a further significant increase in hydroxy conjugated dienes was noted with 18:2, 20:4 and 22:6 isomers being identified (P less than 0.01 vs. end ischemia). By 30 min of reperfusion the concentration of phospholipid oxidation products had returned to pre-ischemic levels. This study presents the first chemically rigorous in vivo evidence for the formation of products of phospholipid oxidation (hydroxy conjugated dienes) during myocardial ischemia and reperfusion and supports the concept of oxygen free radical mediated lipid peroxidation. This study emphasizes the formation of phospholipid oxidation products during ischemia and particularly during the early phase of reperfusion and illustrates the transient nature of these products in myocardial phospholipid.

Quantitation of postischemic skeletal muscle necrosis: Histochemical and radioisotope techniques

Skeletal muscle necrosis will result from prolonged periods of ischemia. The purpose of this study was to develop a method to estimate the extent of necrosis using nitroblue tetrazolium staining and technetium scanning. The bilateral canine gracilis muscle preparation with total vascular isolation was exposed to 4 hr of complete normothermic ischemia followed by reperfusion. After 45 hr of reperfusion 99mTc pyrophosphate (PYP) was injected and 3 hr later the muscles were harvested, cut into six slices, and stained with nitroblue tetrazolium. Biopsies were taken from tetrazolium-positive and -negative areas for electron microscopy to confirm the ability of the stain to distinguish viable from necrotic muscle. Computerized planimetry of the staining pattern was used to estimate the extent of necrosis as a percentage of the total muscle. Electron microscopy confirmed the validity of nitroblue tetrazolium to discriminate between viable and necrotic skeletal muscle in this experimental model. After 4 hr of ischemia the percentage necrosis was 30.2 +/- 6.1% (mean +/- SEM, n = 12), there was no difference in the extent of necrosis in left vs right paired muscles, using tetrazolium staining or technetium PYP uptake. There was a statistically significant correlation between the percentage necrosis and the density of 99mTc PYP uptake per muscle (r = 0.83, P less than 0.001) and per slice (r = 0.94, P less than 0.001). This study demonstrates the ability of tetrazolium staining to accurately differentiate between viable and necrotic skeletal muscle and provides a reproducible method for estimating the extent of necrosis in the gracilis muscle model.

Cell transplantation to prevent heart failure: a comparison of cell types

BACKGROUND Autologous cell transplantation may restore viable muscle after a myocardial infarction. We compared the effect of three cell types or an angiotensin-converting enzyme (ACE) inhibitor on preservation of ventricular function after cardiac injury. METHODS A uniform transmural myocardial scar was created in adult rats by cryoinjury. Three weeks later the rats were randomly assigned to one of four blinded treatments: transplantation with 5 x 10(6) aortic smooth muscle cells (SMC, n = 12), ventricular heart cells (VHC, n = 13), skeletal muscle cells (SKC, n = 13) or culture medium alone (control, n = 11). The ACE inhibitor group (n = 8) received enalapril (1.0 mg/kg per day), also beginning 3 weeks after cryoinjury. Five and 12 weeks after transplantation, left ventricle (LV) function was assessed in a Langendorff apparatus, and histologic and immunohistological evaluation of the LV scars was performed. RESULTS At 5 weeks, greater scar elastin content and better LV function was noted with cell transplantation or ACE inhibitor therapy compared with control rats (p < 0.05). Twelve weeks after transplantation, cell-transplanted rats still had greater elastin content and better LV function than control rats, although elastin content and LV function had declined in ACE inhibitor-treated animals to levels below those observed in control rats (p < 0.05). CONCLUSIONS Transplantation of SMC, VHC, and SKC preserved ventricular function equivalent to the effects of an ACE inhibitor. Muscle cell transplantation, but not ACE inhibitor therapy, continues to be effective later after cryoinjury. No differences were detected between the muscle cells.

Lactate release during reperfusion predicts low cardiac output syndrome after coronary bypass surgery

BACKGROUND Cardioplegic arrest induces anaerobic myocardial metabolism with a net production of lactate from glycolysis. However, persistent lactate release during reperfusion suggests a delayed recovery of normal aerobic metabolism and may lead to depressed myocardial function necessitating inotropic or intraaortic balloon pump support (low output syndrome [LOS]). We examined the relation between perioperative myocardial metabolism and postoperative clinical outcomes in patients undergoing isolated coronary artery bypass surgery (CABG). METHODS We reviewed 623 patients who were enrolled in clinical studies evaluating perioperative myocardial metabolism between 1983 and 1996. Arterial and coronary sinus blood samples were obtained intraoperatively to assess myocardial metabolism. Clinical data regarding patient demographics and postoperative outcomes were prospectively collected and entered into our institutional database. RESULTS Low output syndrome developed in 36 patients (5.8%). Myocardial lactate release was higher in these patients compared with those who did not develop postoperative LOS. Advanced age and poor preoperative left ventricular function were independent predictors of lactate release during reperfusion. Persistent lactate release after 5 minutes of reperfusion was the only independent predictor of postoperative LOS in this low-risk population. CONCLUSIONS Persistent lactate release during reperfusion occurs in a significant proportion of low-risk patients undergoing isolated CABG and is an independent predictor of postoperative low cardiac output syndrome. Persistent lactate release during reperfusion suggests a delayed recovery of aerobic myocardial metabolism and may be related to intraoperative misadventure or inadequate myocardial protection. Myocardial lactate release may be useful as an alternative end-point in clinical trials evaluating perioperative myocardial protection.

Hyperglycemia potentiates the proatherogenic effects of C-reactive protein: reversal with rosiglitazone

Accumulating evidence suggests that C-reactive protein (CRP), at concentrations known to predict diverse vascular insults, directly promotes endothelial cell activation, uncovering a proatherosclerotic and proinflammatory phenotype. In the present study, we hypothesized that (a). hyperglycemia would serve to exaggerate the proatherogenic effects of CRP and (b). the PPARgamma agonist, rosiglitazone would attenuate this effect. Human saphenous vein endothelial cells were studied under the following conditions (n= 10 per group): control, human recombinant CRP (25 microg/ml, 24 h), hyperglycemia (25 mM, 24 h) and hyperglycemia + CRP. In each case, the effects of co-incubation with rosiglitazone (1 microM) were evaluated. Nitric oxide and endothelin-1 release from endothelial cells was quantified, in addition to the expression of adhesion molecules and monocyte chemoattractant chemokine (MCP-1). Incubation of endothelial cells with CRP increased endothelin-1 production, and upregulated adhesion molecule and MCP-1 expression. These proatherogenic effects of CRP were potentiated in the presence of hyperglycemia. CRP also decreased endothelial nitric oxide release, and this effect remained unchanged by hyperglycemia. Importantly, the PPARgamma agonist, rosiglitazone, attenuated the proatherogenic effects of CRP under both basal and hyperglycemic conditions. The direct proatherogenic actions of CRP are exaggerated in the hyperglycemic milieu, and attenuated by rosiglitazone. Elevated CRP levels in patients with diabetes may serve to uncover a severe proatherogenic phenotype.

Effects of anaesthetic induction on myocardial function and metabolism: a comparison of fentanyl, sufentanil and alfentanil

Anaesthetic induction may induce myocardial ischaemia. A prospective randomized trial was instituted to compare the effect on ventricular function and myocardial metabolism of induction with fentanyl (FEN) or its analogues sufentanil (SUF) or alfentanil (ALF) in 96 patients undergoing elective coronary artery bypass grafting (CABG). Haemodynamic, metabolic (coronary sinus oxygen and lactate extraction) and gated ventrìculo graphic measurements were made awake pre-induction (PRE), after induction (IND) and after intubation (INT). Induction was performed with FEN 75 μg · kg-1 SUF 15 μg · kg-1 or ALF 125 μg · kg-1 and metocurine. Fentanyl induction was associated with the greatest stability of mean arterial pressure (MAP), cardiac performance, and systolic function without associated myocardial lactate production. SUF produced the greatest depression of systolic function (p < 0.05) but without haemodynamic instability or myocardial lactate production in all but one patient. Induction with ALF produced the greatest reduction in MAP (p < 0.05) associated with the greatest decrease in diastolic compliance (p < 0.05) and 50 per cent incidence of myocardial lactate production (p < 0.05) with no significant change in coronary blood flow or myocardial oxygen consumption.Résuméľinduction de ľanesthésie peut induire de ľischémie myocardique. Une étude prospective randomisée a été conduite afin de comparer ľeffet sur la fonction ventriculaire et le métabolisme myocardique de ľinduction avec le fentanyl (FEN) ou son analogue sufentanil (SUF) ou ľalfentanil(ALF) chez 96 patients devant subir une chirurgie de pontage aorto coronarien élective. Des mesures hémodynamiques, métaboliques (extraction de lactate ďoxyène du sinus coronaire) ainsi que des mesures de la fonction ventriculaire par des méthodes nucléaires étaient faites avant ľinduction (PRE), aprés ľinduction (IND) et après ľintubation (INT). ľinduction était faite avec du fentanyl 75 μg · kg-1, sufentanil 15 μg · kg-1 et fentanil 125 μg · kg-1 associé à la métocurine. ľinduction avec le fentanyl a amené la plus grande stabilité de la pression artérielle moyenne (MAP), la performance cardiaque, et la fonction systolique sans production de lactate par le myocarde. Le sufentanil a produit la plus grande dépression de la fonction systolique (p < 0.05) sans instabilité hémodynamique ou production de lactate par le myocarde chez tous les patients sauf un. ľinduction avec ľalfentanil a produit la plus grande réduction de la pression artérielle moyenne (p < 0.05) associé avec la plus grande diminution de la compliance diastolique (p < 0.05) et 50 pour cent ďincidence de production de lactate par le mvocarde (p < 0.05) sans changement significatif dans le flot sanguin coronarien ou dans la consommation ďoxygène du myocarde.

Water-soluble antioxidant specificity against free radical injury using cultured human ventricular myocytes and fibroblasts and saphenous vein endothelial cells ☆

To better understand the protective effect of water-soluble antioxidants against free radical injury to the reperfused ischemic myocardium, we studied the antioxidant effectiveness of superoxide dismutase (SOD), catalase, ascorbic acid, and Trolox, a water-soluble analogue of alpha-tocopherol, in protecting cultured adult human ventricular myocytes and fibroblasts and saphenous vein endothelial cells from hypoxanthine-xanthine oxidase generated free radicals. The cells were cultured at oxygen tension to 150 and 40 mmHg. Passage P2 to P4 cells were injured by a hypoxanthine-xanthine oxidase free radical generation system. The time when all the cells became shriveled divided by the cell count expressed in terms of 100,000 cells was used to compare cellular susceptibilities to free radical injury and the relative effectiveness of the antioxidants. Fibroblasts were more resistant to free radical injury than myocytes which were more resistant than endothelial cells, when all three cell types were cultured at the same oxygen tension. Trolox and ascorbic acid were effective antioxidants for myocytes while SOD and catalase were ineffective. SOD and catalase were more effective than ascorbic acid as antioxidants for endothelial cells and fibroblasts, while Trolox was ineffective. In summary, we have shown that each cultured cell type has a different susceptibility to free radical damage and that antioxidants are not effective for all cell types.

Delayed Myocardial Metabolic Recovery After Blood Cardioplegia

Previous studies have demonstrated that both myocardial metabolism and ventricular function were depressed after blood cardioplegic arrest for elective coronary artery bypass grafting. To evaluate the etiology of this metabolic defect, we measured the levels of adenine nucleotides and their precursors in 29 patients undergoing elective coronary revascularization. Myocardial biopsy specimens were obtained at 37 degrees C before cardioplegic arrest, immediately after 74 +/- 4 minutes of cardioplegic arrest, and after 30 minutes of reperfusion. Biopsy specimens were analyzed for levels of adenine nucleotides and their precursors by high-performance liquid chromatography. Adenosine triphosphate concentrations decreased with cardioplegic arrest and with reperfusion. Adenosine monophosphate concentrations increased after cardioplegic arrest and remained nearly twice the initial values after reperfusion. The ratio of adenosine monophosphate to adenosine triphosphate doubled after reperfusion, suggesting defective conversion of adenosine monophosphate to adenosine triphosphate. Levels of adenine nucleotide degradation products (adenosine, inosine, and hypoxanthine) increased after cardioplegia and decreased with reperfusion, suggesting a washout of soluble precursors. This study suggests that improvements in myocardial protection should attempt to stimulate mitochondrial energy production and preserve adenine nucleotide precursors.