Nassrene Y. Elmadhun

The pig as a valuable model for testing the effect of resveratrol to prevent cardiovascular disease

Resveratrol is a naturally occurring polyphenol found in the skin of red grapes, peanuts, and red wine that has been shown to modify many cardiovascular risk factors. Small animal models have been extensively used to investigate cardiovascular disease, but the results often fail to translate in clinical trials. Disease‐specific pig models are emerging as clinically useful tools that may offer insight into cardiovascular disease and the effect of drugs such as resveratrol on cardiovascular health. In this paper, we discuss the advantage of using clinically relevant pig models of diabetes, hypercholesterolemia, and myocardial ischemia to investigate the role of resveratrol in cardiovascular disease prevention.

Resveratrol regulates autophagy signaling in chronically ischemic myocardium

OBJECTIVE Autophagy is a cellular process by which damaged components are removed. Although autophagy can result in cell death, when optimally regulated, it might be cardioprotective. Resveratrol is a naturally occurring polyphenol also believed to be cardioprotective. Using a clinically relevant swine model of metabolic syndrome, we investigated the effects of resveratrol on autophagy in the chronically ischemic myocardium. METHODS Yorkshire swine were fed a regular diet (n = 7), a high cholesterol diet (n = 7), or a high cholesterol diet with supplemental resveratrol (n = 6). After 4 weeks, an ameroid constrictor was surgically placed on the left circumflex artery to induce chronic myocardial ischemia. The diets were continued another 7 weeks, and then the ischemic and nonischemic myocardium were harvested for protein analysis. RESULTS In the ischemic myocardium, a high cholesterol diet partly attenuated the autophagy, as determined by an increase in phosphorylated mammalian target of rapamycin (p-mTOR) and a decrease in p70 S6 kinase (P70S6K), lysosome-associated membrane protein (LAMP)-2, and autophagy-related gene 12-5 conjugate (ATG 12-5; P < .05). The addition of resveratrol blunted many of these changes, because the p-mTOR, P70S6K, and LAMP-2 levels were not significantly altered from those of the pigs fed a regular diet. Other autophagy markers were increased with a high cholesterol diet, including light chain 3A-II and beclin 1 (P < .05). In the nonischemic myocardium, beclin 1 was decreased in the high cholesterol-fed pigs (P < .05); otherwise no significant changes in protein expression were noted among the 3 groups. CONCLUSIONS In the chronically ischemic myocardium, resveratrol partly reversed the effects of a high cholesterol diet on autophagy. This might be a mechanism by which resveratrol exerts its cardioprotective effects.

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.

Calpain inhibition decreases myocardial apoptosis in a swine model of chronic myocardial ischemia

INTRODUCTION Calpain is a family of cysteine proteases that has an important role in the initiation, regulation, and execution of cell death. Our recent studies using a hypercholesterolemic swine model demonstrated that in the setting of the metabolic syndrome, calpain inhibition (CI) improved collateral-dependent perfusion and increased expression of proteins implicated in angiogenesis and vasodilation. In this study, we hypothesized that CI (by MLD28170) would decrease myocardial apoptosis in the same model. METHODS Yorkshire swine, all fed a high-cholesterol diet for 4 weeks underwent placement of an ameroid constrictor on the left circumflex coronary artery. Three weeks later, animals received either no drug, termed the high-cholesterol control group (HCC; n = 8); low-dose CI (0.12 mg/kg; LCI, n = 9); or high-dose CI (0.25 mg/kg; HCI, n = 8). The high-cholesterol diet and the CI were continued for 5 weeks, after which the pig was humanely killed and the left ventricular myocardium was harvested and analyzed via terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining, oxyblot analysis, and Western blots. Data were analyzed using the Kruskal-Wallis test. RESULTS The percentage of apoptotic cells to total cells in ischemic myocardial territory was decreased in the LCI and HCI groups compared with the HCC group as shown by TUNEL staining (P = .018). There was a decrease in proapoptotic proteins, including cleaved caspase 3, caspase 9, cleaved caspase 9, Bax, BAD, p-BAD, and Erk 1/2 (P ≤ .049 each), but no decrease in caspase 3 (P = .737). There was also an increase in antiapoptotic proteins, including BCL-2 and p-BCL2 (P ≤ .025 each). In the ischemic myocardium, several proangiogenic proteins were increased in the LCI and HCI groups compared with the HCC group, including p-AKT, p-eNOS, and eNOS (P ≤ .006 each) but there was no increase in AKT (P = .311). CI decreased tissue oxidative stress in both the LCI and HCI groups compared to the HCC group as shown by oxyblot analysis (P = .021). CONCLUSION In the setting of hypercholesterolemia, CI decreases apoptosis and the expression of proteins in proapoptotic signaling pathways. CI also increased expression of proteins implicated in anti apoptotic pathways and improves oxidative stress in ischemic myocardial tissue.

Metformin mitigates apoptosis in ischemic myocardium.

BACKGROUND Epidemiologic data has shown that metformin confers a survival advantage in patients with cardiovascular disease. Although the underlying cardioprotective mechanism is unclear, it appears to be independent of metformins insulin-sensitizing effect. The purpose of this study was to evaluate the effect of metformin on the apoptosis pathway in the ischemic and nonischemic cardiac tissue in a swine model of metabolic syndrome. MATERIALS AND METHODS Ossabaw miniswine were fed either a regular diet (Ossabaw control, n = 8), a high-cholesterol diet (Ossabaw high cholesterol, n = 8), or a high-cholesterol diet supplemented with metformin (Ossabaw high-cholesterol metformin, n = 8). After 9 wk, all animals underwent placement of an ameroid constrictor to the left circumflex coronary artery to induce chronic ischemia. Seven weeks after ameroid placement, animals underwent cardiac harvest. RESULTS In the chronically ischemic myocardium, metformin significantly upregulates prosurvival proteins: extracellular signal-regulated kinases, nuclear factor κB, phosphorylated endothelial nitric oxide synthase, and P38. Metformin also significantly inhibits or downregulates proapoptosis proteins: FOXO3 and caspase 3. Metformin decreased the percent apoptotic cells in the ischemic and nonischemic myocardium. There was no difference in arteriolar density, capillary density, intramyocardial fibrosis, or collagen deposition in the ischemic or nonischemic myocardium. CONCLUSIONS Metformin selectively alters the apoptosis pathway by inhibiting FOXO3 and decreasing the active form of caspase 3, cleaved caspase 3. Metformin also upregulates mitogen-activated kinase proteins p38 and extracellular signal-regulated protein kinases 1 and 2, which are considered cardioprotective during ischemic preconditioning. Perhaps, the altered activation of the apoptosis pathway in ischemic myocardium is one mechanism by which metformin is cardioprotective.

Calpain inhibition improves collateral-dependent perfusion in a hypercholesterolemic swine model of chronic myocardial ischemia

PURPOSE Calpain overexpression is implicated in aberrant angiogenesis. We hypothesized that calpain inhibition (MDL28170) would improve collateral perfusion in a swine model with hypercholesterolemia and chronic myocardial ischemia. METHODS Yorkshire swine fed a high cholesterol diet for 4 weeks underwent surgical placement of an ameroid constrictor to their left circumflex coronary artery. Three weeks later, animals received no drug, high cholesterol control group (n = 8); low-dose calpain inhibition (0.12 mg/kg; n = 9); or high-dose calpain inhibition (0.25 mg/kg; n = 8). The heart was harvested after 5 weeks. RESULTS Myocardial perfusion in ischemic myocardium significantly improved with high-dose calpain inhibition at rest and with demand pacing (P = .016 and .011). Endothelium-dependent microvessel relaxation was significantly improved with low-dose calpain inhibition (P = .001). There was a significant increase in capillary density, with low-dose calpain inhibition and high-dose calpain inhibition (P = .01 and .01), and arteriolar density with low-dose calpain inhibition (P = .001). Calpain inhibition significantly increased several proangiogenic proteins, including vascular endothelial growth factor (P = .02), vascular endothelial growth factor receptor 1 (P = .003), vascular endothelial growth factor receptor 2 (P = .003), and talin, a microvascular structural protein (P = .0002). There was a slight increase in proteins implicated in endothelial-dependent (nitric oxide mediated) relaxation, including extracellular signal-regulated kinase, phosphorylated extracellular signal-regulated kinase, and inducible nitric oxide synthase with calpain inhibition. CONCLUSIONS In the setting of hypercholesterolemia, calpain inhibition improved perfusion, with a trend toward increased collateralization on angiography and increased capillary and arteriolar densities in ischemic myocardium. Calpain inhibition also improved endothelium-dependent microvessel relaxation and increased expression of proteins implicated in angiogenesis and vasodilatation.

Differential effects of atorvastatin on autophagy in ischemic and nonischemic myocardium in Ossabaw swine with metabolic syndrome

OBJECTIVES The perioperative administration of pleomorphic statin drugs has been implicated in improving outcomes after cardiac surgery. Adaptive autophagy is a highly conserved cellular process that allows for the elimination of dysfunctional cell components in response to stress and survival under starving conditions. We sought to investigate the effects of the statin drug atorvastatin on autophagy in ischemic and nonischemic myocardia using a clinically relevant porcine model of metabolic syndrome. METHODS Male Ossabaw swine were fed a regular diet (n = 8), a high-cholesterol diet (n = 8), or a high-cholesterol diet with supplemental atorvastatin (1.5 mg/kg/d) (n = 8). After 14 weeks, all animals underwent surgical placement of an ameroid constrictor to the circumflex coronary artery to induce chronic ischemia. Nonischemic and ischemic myocardia were harvested 6 months after initiation of the diet and processed for Western blotting. RESULTS In the nonischemic myocardium, Western blot results demonstrate that a high cholesterol diet resulted in a statistically significant decrease in autophagy as indicated by an increase in mammalian target of rapamycin and the accumulation of several essential autophagy markers, including Beclin-1, light chain 3B-I, and light chain 3B-II. Atorvastatin supplementation prevented these changes and resulted in an increase in autophagy as indicated by a decrease in autophagy flux marker P62. In the ischemic myocardium, atorvastatin had the opposite effect, with a decrease in autophagy flux as indicated by an increase in p62 and an accumulation of light chain 3B-I, light chain B-II, and lysosome-associated membrane protein 2. CONCLUSIONS Atorvastatin administration has differential effects on autophagy in ischemic and nonischemic myocardia. In the setting of metabolic syndrome, atorvastatin stimulates autophagy in nonischemic myocardium while partly inhibiting autophagy in ischemic myocardium. The differential regulation on autophagy may, in part, explain the cardioprotective effect of statins in both ischemic and nonischemic myocardia, and these findings may have implications in the setting of cardiac surgery.

Metabolic syndrome impairs notch signaling and promotes apoptosis in chronically ischemic myocardium

OBJECTIVE Impaired angiogenesis is a known consequence of metabolic syndrome (MetS); however, the mechanism is not fully understood. Recent studies have shown that the notch signaling pathway is an integral component of cardiac angiogenesis. We tested, in a clinically relevant swine model, the effects of MetS on notch and apoptosis signaling in chronically ischemic myocardium. METHODS Ossabaw swine were fed either a regular diet (control [CTL], n = 8) or a high-cholesterol diet (MetS, n = 8) to induce MetS. An ameroid constrictor was placed to induce chronic myocardial ischemia. Eleven weeks later, the wine underwent cardiac harvest of the ischemic myocardium. RESULTS Downregulation of pro-angiogenesis proteins notch2, notch4, jagged2, angiopoietin 1, and endothelial nitric oxide synthase were found in the MetS group compared with the CTL group. Also, upregulation of pro-apoptosis protein caspase 8 and downregulation of anti-angiogenesis protein phosphorylated forkhead box transcription factor 03 and pro-survival proteins phosphorylated P38 and heat shock protein 90 were present in the MetS group. Cell death was increased in the MetS group compared with the CTL group. Both CTL and MetS groups had a similar arteriolar count and capillary density, and notch3 and jagged1 were both similarly concentrated in the smooth muscle wall. CONCLUSIONS MetS in chronic myocardial ischemia significantly impairs notch signaling by downregulating notch receptors, ligands, and pro-angiogenesis proteins. MetS also increases apoptosis signaling, decreases survival signaling, and increases cell death in chronically ischemic myocardium. Although short-term angiogenesis appears unaffected in this model of early MetS, the molecular signals for angiogenesis are impaired, suggesting that inhibition of notch signaling might underlie the decreased angiogenesis in later stages of MetS.

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.

Arterial territory-specific phosphorylated retinoblastoma protein species and CDK2 promote differences in the vascular smooth muscle cell response to mitogens

Despite recent advances in medical procedures, cardiovascular disease remains a clinical challenge and the leading cause of mortality in the western world. The condition causes progressive smooth muscle cell (SMC) dedifferentiation, proliferation, and migration that contribute to vascular restenosis. The incidence of disease of the internal mammary artery (IMA), however, is much lower than in nearly all other arteries. The etiology of this IMA disease resistance is not well understood. Here, using paired primary IMA and coronary artery SMCs, serum stimulation, siRNA knockdowns, and verifications in porcine vessels in vivo, we investigate the molecular mechanisms that could account for this increased disease resistance of internal mammary SMCs. We show that the residue-specific phosphorylation profile of the retinoblastoma tumor suppressor protein (Rb) appears to differ significantly between IMA and coronary artery SMCs in cultured human cells. We also report that the differential profile of Rb phosphorylation may follow as a consequence of differences in the content of cyclin-dependent kinase 2 (CDK2) and the CDK4 phosphorylation inhibitor p15. Finally, we present evidence that siRNA-mediated CDK2 knockdown alters the profile of Rb phosphorylation in coronary artery SMCs, as well as the proliferative response of these cells to mitogenic stimulation. The intrinsic functional and protein composition specificity of the SMCs population in the coronary artery may contribute to the increased prevalence of restenosis and atherosclerosis in the coronary arteries as compared with the internal mammary arteries.