Thomas A. Burgess

Resveratrol Preserves Myocardial Function and Perfusion in Remote Nonischemic Myocardium in a Swine Model of Metabolic Syndrome

BACKGROUND Resveratrol has been shown to reverse some of the detrimental effects of metabolic syndrome (MetS). We sought to define the impact of supplemental resveratrol on normal myocardium remote from an ischemic territory in a swine model of MetS and chronic myocardial ischemia. STUDY DESIGN Yorkshire swine were fed a normal diet (control), a high cholesterol diet (HCD), or a high cholesterol diet with orally supplemented resveratrol (HCD-R; 100 mg/kg/day). Four weeks after diet modification, myocardial ischemia was induced by ameroid constrictor placement. Seven weeks later, myocardial tissue from a territory remote from the ischemia was harvested. Animals in the HCD and HCD-R groups underwent functional cardiac MRI before ischemia and before sacrifice. Tissue was harvested for protein expression analysis. RESULTS After 7 weeks of ischemia, regional left ventricular systolic function was significantly increased in HCD-R as compared with HCD animals. During ventricular pacing the HCD group had significantly decreased flow (p = 0.03); perfusion in the HCD-R was preserved as compared with the control. There was no difference in microvascular relaxation. Expression of metabolic proteins Sirt-1 (p = 0.002), AMPkinase (p = 0.02), and carnitine palmitoyltransferase-I (p = 0.002) were upregulated in the HCD-R group. Levels of protein oxidative stress were significantly increased in the HCD and HCD-R groups, as compared with the controls (p = 0.003). Activated endothelial nitric oxide synthase (eNOS) was increased in the HCD-R group (p = 0.01). There was no difference in myocardial endothelial cell density between the groups; however, dividing endothelial cells were decreased in the HCD and HCD-R groups (p = 0.006). CONCLUSIONS Resveratrol supplementation improves regional left ventricular function and preserves perfusion to myocardium remote from an area of ischemia in an animal model of metabolic syndrome and chronic myocardial ischemia.

Effects of Red Wine and Vodka on Collateral-Dependent Perfusion and Cardiovascular Function in Hypercholesterolemic Swine

Background— Moderate consumption of alcohol, particularly red wine, has been shown to decrease cardiac risk. We used a hypercholesterolemic swine model of chronic ischemia to examine the effects of 2 alcoholic beverages on the heart. Methods and Results— Yorkshire swine fed a high-cholesterol diet underwent left circumflex ameroid constrictor placement to induce chronic ischemia at 8 weeks of age. One group (HCC, n=9) continued on the diet alone, the second (HCW, n=8) was supplemented with red wine (pinot noir, 12.5% alcohol, 375 mL daily), and the third (HCV, n=9) was supplemented with vodka (40% alcohol, 112 mL daily). After 7 weeks, cardiac function was measured, and ischemic myocardium was harvested for analysis of perfusion, myocardial fibrosis, vessel function, protein expression, oxidative stress, and capillary density. Platelet function was measured by aggregometry. Perfusion to the ischemic territory as measured by microsphere injection was significantly increased in both HCW and HCV compared with HCC at rest, but in only the HCW group under ventricular pacing. Microvessel relaxation response to adenosine 5′-diphosphate was improved in the HCW group alone as was regional contractility in the ischemic territory, although myocardial fibrosis was decreased in both HCW and HCV. Expression of proangiogenic proteins phospho-endothelial nitric oxide synthase and vascular endothelial growth factor was increased in both HCW and HCV, whereas phospho-mammalian target of rapamycin was increased only in the HCV group. Expression of Sirt-1 and downstream antioxidant phospho-FoxO1 was increased only in the HCW group. Protein oxidative stress was decreased in the HCW group alone, whereas capillary density was increased only in the HCV group. There was no significant difference in platelet function between groups. Conclusion— Moderate consumption of red wine and vodka may reduce cardiovascular risk by improving collateral-dependent perfusion through different mechanisms. Red wine may offer increased cardioprotection related to its antioxidant properties.

Cardioprotective effects of red wine and vodka in a model of endothelial dysfunction.

BACKGROUND Moderate alcohol consumption is largely believed to be cardioprotective, while red wine is hypothesized to offer benefit in part due to the proangiogenic and antioxidant properties of polyphenols. We investigated the cardiovascular effects of both red wine and vodka in a swine model of endothelial dysfunction. METHODS Twenty-seven male Yorkshire swine fed a high-fat/cholesterol diet were divided into three groups and received either no alcohol (Control), red wine, or vodka. After 7 wk, myocardial perfusion was measured, and ventricular tissue was analyzed for microvascular reactivity and immunohistochemical studies. RESULTS There were no differences in myocardial perfusion, in arteriolar or capillary density, or in VEGF expression among groups. Total protein oxidation as well as expression of superoxide dismutase-1 and -2 and NADPH oxidase was decreased in both treatment groups compared to controls. Endothelium-dependent microvessel relaxation, however, was significantly improved only in the red wine-supplemented group. CONCLUSIONS Supplementation with both red wine and vodka decreased oxidative stress by several measures, implicating the effects of ethanol in reducing oxidative stress in the myocardium. However, it was only in the red wine-supplemented group that an improvement in microvessel function was observed. This suggests that a component of red wine, independent of ethanol, possibly a polyphenol such as resveratrol, may confer cardioprotection by normalizing endothelial dysfunction induced by an atherogenic diet.

Effects of selective cyclooxygenase-2 and nonselective cyclooxygenase inhibition on myocardial function and perfusion.

Nonselective nonsteroidal anti-inflammatory drugs and selective cyclooxygenase-2 (COX-2) inhibitors are purported to increase adverse cardiovascular events. We hypothesized that COX-2 inhibitors would alter myocardial blood flow, microvascular reactivity, oxidative stress, and prostaglandin levels. Adult Yorkshire swine were divided into 3 groups: no drug (control, n = 7), a nonselective COX inhibitor (naproxen 400 mg daily, NAP, n = 7), or a selective COX-2 inhibitor (celecoxib 200 mg daily, CBX, n = 7). After 7 weeks, physiologic measurements were taken and tissue harvested. Animals in the CBX group demonstrated significantly higher blood pressure and rate-pressure product. The NAP and CBX groups demonstrated an increased microvascular contraction response to serotonin. The NAP group showed increased myocardial levels of thromboxane and lower levels of prostacyclin. Levels of protein oxidative stress were increased in the CBX group. Myocardial apoptosis was lowest in the NAP group. Immunoblotting demonstrated decreased vascular endothelial growth factor and phosphorylated endothelial nitric oxide synthase expression in the NAP and CBX groups. Myocardial tumor necrosis factor-α was increased in both treated groups. Immunostaining for thromboxane A2 synthase and receptor demonstrated expression within the vascular smooth muscle and no observable differences between groups. Nonselective and selective COX inhibition does not alter myocardial perfusion but results in altered myocardial and vascular physiology that may have implications regarding cardiovascular risk.

Resveratrol supplementation abrogates pro-arteriogenic effects of intramyocardial vascular endothelial growth factor in a hypercholesterolemic swine model of chronic ischemia.

BACKGROUND Clinical trials of therapeutic angiogenesis with vascular endothelial growth factor (VEGF) have been disappointing, owing likely to endothelial dysfunction. We used a swine model of chronic ischemia and endothelial dysfunction to determine whether resveratrol coadministration would improve the angiogenic response to VEGF therapy. METHODS Yorkshire swine fed a high-cholesterol diet underwent left circumflex ameroid constrictor placement, and were given either no drug (high cholesterol control [HCC], n = 8), perivascular VEGF (2 μg sustained release [high cholesterol VEGF-treated; HCV], n = 8), or VEGF plus oral resveratrol (10 mg/kg, [high cholesterol VEGF- and resveratrol-treated; HCVR], n = 8). After 7 weeks, myocardial contractility, perfusion, and microvessel reactivity in the ischemic territory were assessed. Tissue was analyzed for vessel density, oxidative stress, and protein expression. RESULTS Myocardial perfusion was significantly improved in the HCV group compared with the HCC group; resveratrol coadministration abrogated this improvement. There were no differences in regional myocardial contractility between groups. Endothelium-dependent microvessel relaxation was improved in the HCVR group, and endothelium-independent relaxation response was similar between groups. Arteriolar density was greatest in the HCV group, whereas capillary density was similar between groups. Expression of Akt and phospho-endothelial nitric oxide synthase were increased in the HCVR group. Total protein oxidative stress and myeloperoxidase expression were reduced in the HCVR group, but so was the oxidative-stress dependent phosphorylation of vascular endothelial cadherin (VE-cadherin) and β-catenin. CONCLUSION Although resveratrol coadministration decreases oxidative stress and improves endothelial function, it abolishes improvements in myocardial perfusion and arteriolar density afforded by VEGF treatment alone. This effect is due likely to inhibition of the oxidative stress-dependent phosphorylation of VE-cadherin, an essential step in the initiation of arteriogenesis.

Alcohol consumption improves insulin signaling in the myocardium.

BACKGROUND In a previous study, we demonstrated that swine with metabolic syndrome treated with alcohol had improved insulin signaling. We developed a follow-up study to evaluate the effects of alcohol on ischemic myocardium in animals without metabolic syndrome. METHODS Fourteen Yorkshire swine underwent placement of an ameroid constrictor to induce chronic myocardial ischemia. Postoperatively, one group was supplemented with ethanol (ETOH), and one group was supplemented with sucrose (SUC) daily to normalize caloric intake. After 7 weeks, all animals underwent dextrose challenge and harvest of nonischemic and ischemic myocardium. Tissues were analyzed for protein expression and histologic analysis. RESULTS There was no difference in body mass index, serum glucose or insulin levels. However, ethanol supplementation up-regulated phosphoinostitide 3-kinase, phosphorylated protein kinase B, protein kinase B, and phosphorylated Forkhead Box 01 expression, which may promote insulin signaling, and down-regulated inhibitors of insulin signaling pIRS1 and pIRS2. There was no difference in intramyocardial glycogen but there was increased GLUT4 expression in the ETOH group, which may promote glucose use. CONCLUSION Despite similar serum glucose and insulin levels, alcohol consumption up-regulates the insulin signaling pathway in the absence of metabolic syndrome in both nonischemic and chronically ischemic myocardium. These results suggest that alcohol selectively up-regulates the insulin signaling pathway despite normoglycemia.

Ethanol Promotes Arteriogenesis and Restores Perfusion to Chronically Ischemic Myocardium

Background— Moderate alcohol consumption is known to be cardioprotective compared with either heavy drinking or complete abstinence. We assessed the hypothesis that ethanol supplementation would improve myocardial function in the setting of chronic ischemia. Methods and Results— Sixteen male Yorkshire swine underwent placement of an ameroid constrictor into the left circumflex artery to induce chronic myocardial ischemia. Postoperatively, animals were supplemented with either 90 mL of ethanol (EtOH) daily (50%/V, EtOH) or 80 g of sucrose of equal caloric value (SUC), serving as controls. Seven weeks after ameroid placement, arteriolar density (1.74±0.210% versus 3.11±0.368% area of arterioles per low-powered field in sucrose (SUC) versus EtOH; P=0.004), myocardial perfusion (ratio of blood flow to the at-risk myocardium compared with the normal ventricle during demand pacing was 0.585±0.107 versus 1.08±0.138 for SUC versus EtOH; P=0.014), and microvascular reactivity were significantly increased in ethanol-treated animals compared with controls in the at-risk myocardium. Analysis of vascular endothelial growth factor and NOTCH pathway signaling suggested proneovascular and proliferative activity in the ischemic area. The average peak blood alcohol level in the treatment group was 40±4 mg/dL, consistent with levels of moderate drinking in humans. Conclusions— Ethanol supplementation increased arteriolar density and significantly improved myocardial perfusion and endothelium-dependent vasorelaxation in chronically ischemic myocardium. These findings suggest that, at moderate doses, ethanol directly promotes vasculogenesis and improves microvascular function, resulting in significant improvements in myocardial perfusion in the setting of chronic ischemia.

Effects of cyclooxygenase inhibition on cardiovascular function in a hypercholesterolemic swine model of chronic ischemia

The cardiovascular effects of cyclooxygenase (COX) inhibition remain controversial, especially in the setting of cardiovascular comorbidities. We examined the effects of nonselective and selective COX inhibition on cardiovascular function in a hypercholesterolemic swine model of chronic ischemia. Twenty-four intact male Yorkshire swine underwent left circumflex ameroid constrictor placement and were subsequently given either no drug (HCC; n = 8), a nonselective COX inhibitor (440 mg/day naproxen; HCNS; n = 8), or a selective COX-2 inhibitor (200 mg/day celecoxib; HCCX; n = 8). After 7 wk, myocardial functional was measured and myocardium from the nonischemic ventricle and ischemic area-at-risk (AAR) were analyzed. Regional function as measured by segmental shortening was improved in the AAR of HCCX compared with HCC. There was no significant difference in perfusion to the nonischemic ventricle between groups, but myocardial perfusion in the AAR was significantly improved in the HCCX group compared with controls at rest and during pacing. Endothelium-dependent microvessel relaxation was diminished by ischemia in HCC animals, but both naproxen and celecoxib improved vessel relaxation in the AAR compared with controls, and also decreased the vasoconstrictive response to serotonin. Thromboxane levels in the AAR were decreased in both HCNS and HCCX compared with HCC, whereas prostacyclin levels were decreased only in HCNS, corresponding to a decrease in prostacyclin synthase expression. Chronic ischemia increased apoptosis in Troponin T negative cells and intramyocardial fibrosis, both of which were reduced by celecoxib administration in the AAR. Capillary density was decreased in both the HCNS and HCCX groups. Protein oxidative stress was decreased in both HCNS and HCCX, whereas lipid oxidative stress was decreased only in the HCCX group. Thus nonselective and especially selective COX inhibition may have beneficial myocardial effects in the setting of hypercholesterolemia and chronic ischemia. Whether these effects modulate cardiovascular risk in patients taking these drugs remains to be seen, but evidence to date suggests that they do not.

Temporal and Spatial Changes in Collateral Formation and Function During Chronic Myocardial Ischemia

BACKGROUND We investigated time dependence and spatial progression of cardiac function and angiogenesis signaling in a porcine model of chronic myocardial ischemia. STUDY DESIGN Yorkshire mini-swine (n = 7/group) were subjected to chronic myocardial ischemia by placing an ameroid constrictor on the left circumflex coronary artery under general anesthesia. Swine were sacrificed after either 4 or 7 weeks of ischemia. Myocardial function, angiographic evidence of angiogenesis, microvessel function, molecular signaling, and levels of apoptosis and oxidative stress were assessed. RESULTS Flow reserve was significantly increased at 7 versus 4 weeks. Myocardial function (+dP/dt) improved 1.5-fold by 7 weeks. In the ischemic territory, microvessels at 4 weeks displayed abnormal contraction responses to serotonin, which diminished at 7 weeks. Delta-like ligand 4 protein expression decreased at 7 weeks; expression of vascular endothelial growth factor (VEGF) and phospho-endothelial nitric acid synthase (eNOS) increased. The number of apoptotic cells was decreased at 7 weeks, and antiapoptotic markers heat shock protein (HSP) 27 and HSP 90 were upregulated at 7 weeks. There was an increase in proliferating endothelial cells at 7 weeks as compared with 4 weeks. In the adjacent normal ventricle, microvessels demonstrated smaller contraction responses to endothelin-1 and serotonin at 7 weeks. There was an increase in protein peroxidation in the ischemic territory at 7 weeks. CONCLUSIONS Over time, myocardial perfusion, function, and angiogenic signaling improved in the ischemic myocardium and adjacent normal territory compared with what is observed shortly after coronary occlusion.

Effect of Dimerized Thrombin Fragment TP508 on Acute Myocardial Ischemia Reperfusion Injury in Hypercholesterolemic Swine

The thrombin-related peptide TP508 is a 23-amino acid monomer that represents a portion of the receptor binding domain in the thrombin molecule. TP508 is also known to readily convert to a dimer in an aqueous environment. In this study the dimeric form of TP508 was investigated in a porcine model of acute myocardial ischemia reperfusion injury (and compared with its monomer). Twenty-four hypercholesterolemic pigs underwent 60 min of mid-left anterior descending coronary artery occlusion followed by 120 min of reperfusion and received either vehicle (n = 6), TP508 monomer (n = 6), or two different doses of dimer (n = 6). Infarct size was significantly reduced in the monomer and two dimer groups compared with vehicle. Improvement in both endothelium-dependent and -independent coronary microvascular relaxations was also observed in treated groups. In addition, the expression of 27-kDa heat shock protein, αB-crystalline, and phosphorylated B-cell lymphoma 2 (Ser70) in the ischemic area at risk were higher in treated groups than in vehicle, whereas the expression of cleaved poly-ADP ribose polymerase was lower in treated groups. Finally, there were fewer apoptotic cells in treated groups than in vehicle. This study suggests that TP508 dimer provides a myocardial-protective effect on acute ischemia reperfusion injury in hypercholesterolemic swine, similar to TP508 monomer, by up-regulating cell survival pathways or down-regulating apoptotic pathways.